US20040152174A1 - Process for the biological production of 1,3-propanediol with high yield - Google Patents

Process for the biological production of 1,3-propanediol with high yield Download PDF

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US20040152174A1
US20040152174A1 US10/680,286 US68028603A US2004152174A1 US 20040152174 A1 US20040152174 A1 US 20040152174A1 US 68028603 A US68028603 A US 68028603A US 2004152174 A1 US2004152174 A1 US 2004152174A1
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gene
propanediol
expression
glycerol
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Marguerite Cervin
Philippe Soucaille
Fernando Valle
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Danisco US Inc
Nutrition and Biosciences USA 4 Inc
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C12P7/20—Glycerol

Definitions

  • This invention comprises a process for the bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism.
  • 1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
  • ethylene oxide may be converted to 1,3-propanediol over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid, by the catalytic solution phase hydration of acrolein followed by reduction, or from compounds such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from group VIII of the periodic table.
  • phosphine phosphine
  • water carbon monoxide
  • hydrogen and an acid by the catalytic solution phase hydration of acrolein followed by reduction, or from compounds such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from group VIII of the periodic table.
  • 1,3-propanediol can be produced from the fermentation of glycerol.
  • Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter.
  • glycerol is converted to 1,3-propanediol in a two step, enzyme catalyzed reaction sequence.
  • a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water, Equation 1.
  • 3-HPA is reduced to 1,3-propanediol by a NAD + -linked oxidoreductase, Equation 2.
  • the 1,3-propanediol is not metabolized further and, as a result,
  • NADH reduced ⁇ -nicotinamide adenine dinucleotide
  • NAD+ nicotinamide adenine dinucleotide
  • the specific 1,3-propanediol oxidoreductases (dhaTs) of these microorganisms are known to belong to the family of type III alcohol dehydrogenases; each exhibits a conserved iron-binding motif and has a preference for the NAD + /NADH linked interconversion of 1,3-propanediol and 3-HPA.
  • the NAD + /NADH linked interconversion of 1,3-propanediol and 3-HPA is also catalyzed by alcohol dehydrogenases which are not specifically linked to dehydratase enzymes (for example, horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)), albeit with less efficient kinetic parameters.
  • Glycerol dehydratase (E.C. 4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinct enzymes that are encoded by distinct genes.
  • Diol dehydratase genes from Klebsiella oxytoca and Salmonella typhimurium are similar to glycerol dehydratase genes and are clustered in a group which comprises genes analogous to orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999); Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); GenBank AF026270).
  • dhaB glycerol dehydratase
  • dhaT 1,3-propanediol oxidoreductase
  • dhaD glycerol dehydrogenase
  • dhaK dihydroxyacetone kinase
  • the dha regulon, in Klebsiella pneumoniae and Citrobacter freundii also encompasses a gene encoding a transcriptional activator protein (dhaR).
  • dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.
  • glycerol Biological processes for the preparation of glycerol are known.
  • the overwhelming majority of glycerol producers are yeasts but some bacteria, other fungi, and algae are also known. Both bacteria and yeasts produce glycerol by converting glucose or other carbohydrates through the fructose-1,6-bisphosphate pathway in glycolysis or the Embden Meyerhof Parnas pathway.
  • Dihydroxyacetone phosphate is converted to glycerol-3-phosphate by the action of glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphate is converted to glycerol by the action of glycerol-3-phosphatase.
  • DAR1, GPD1 glycerol-3-phosphate dehydrogenase
  • S. diastaticus Wang et al., J. Bact. 176, 7091-7095 (1994)
  • the DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al. (supra) recognize that DAR1 is regulated by the cellular osmotic environment but do not suggest how the gene might be used to enhance 1,3-propanediol production in a recombinant microorganism.
  • glycerol-3-phosphate dehydrogenase enzymes have been isolated: for example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from Saccharomyces cerevisiae (Larason et al., Mol. Microbiol. 10, 1101 (1993)) and Albertyn et al. ( Mol. Cell. Biol. 14, 4135 (1994)) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae . Like Wang et al. (supra), both Albertyn et al. and Larason et al. recognize the osmosensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of 1,3-propanediol in a recombinant microorganism.
  • G3PDH glycerol-3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)). Like the genes encoding G3PDH, it appears that GPP2 is osmosensitive.
  • WO 9634961 and Hernandez-Montalvo et al. describe E. coli strains that have “PTS” minus/glucose plus phenotypes.
  • EP 1170376 A1 discloses deletion of a gene for NADH dehydratase II to improve energy efficiency.
  • WO 2001016346 describes the utility of “aldehyde dehydrogenase A” and “aldehyde dehydrogenase B” for the production of 3-hydroxypropionic acid.
  • WO 9635796 U.S. Pat. No. 5,686,276, E. I. du Pont de Nemours and Company (“DuPont”) discloses a method for the production of 1,3-propanediol from a carbon substrate other than glycerol or dihydroxyacetone (especially, e.g., glucose), using a single microorganism comprising a dehydratase activity.
  • WO 9928480 discloses a similar method with advantages derived from expressing exogenous activities of one or both of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase while disrupting one or both of endogenous activities glycerol kinase and glycerol dehydrogenase.
  • WO 9821339 U.S. Pat. No.
  • DuPont discloses a method for the production of 1,3-propanediol comprising a recombinant microorganism further comprising a dehydratase and protein X (later identified as being a dehydratase reactivation factor peptide).
  • WO 2001012833 (DuPont) describes an improvement to the process where a significant increase in titer (grams product per liter) is obtained by virtue of a non-specific catalytic activity (distinguished from 1,3-propanediol oxidoreductase encoded by dhaT) to convert 3-hydroxypropionaldehyde to 1,3-propanediol.
  • glycerol as an intermediate substrate for a two-step sequential reaction in which a dehydratase enzyme (typically a coenzyme B 12 -dependent dehydratase) converts glycerol to 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH- (or NADPH-) dependent oxidoreductase.
  • a dehydratase enzyme typically a coenzyme B 12 -dependent dehydratase
  • NADH- (or NADPH-) dependent oxidoreductase The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence for the production of 1,3-propanediol.
  • the present invention provides for bioconverting a fermentable carbon source to 1,3-propanediol at higher yield than previously obtained and with the use of a single microorganism.
  • the yield obtained is greater than 35%, and preferably greater than 40%.
  • Glucose is used as a model substrate and Escherichia coli is used as the model host microorganism with the useful genetic modifications and disruptions detailed herein.
  • E. coli strain comprising:
  • disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprises one or more of
  • the E. coli embodiments described above can further comprise one or more of
  • E. coli embodiments described above can further comprise one or more of
  • E. coli embodiments described above can further comprise one or more of:
  • 1,3-propanediol can be bioproduced by contacting an E. coli strain described herein with a suitable carbon substrate such as glucose under suitable conditions for fermentation.
  • 1,3-propanediol can be bioproduced by contacting an E. coli strain described herein, the E. coli strain further comprising an active:
  • any of the embodiments disclosed above may also include the constructs pSYCO101, pSYCO103, pSYCO106, pSYCO109 or their corresponding nucleotide sequences SEQ ID NOs:65, 66, 67, or 68.
  • FIG. 1 shows 1,3-propanediol production compared as between two fermentations run essentially as described in GENERAL METHODS.
  • the strain used was FMP′::Km/pSYCO103.
  • the strain used was FMP/pSYCO103.
  • Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Res. 13, 3021-3030 (1985) and in the Biochemical Journal 219, 345-373 (1984) which are herein incorporated by reference.
  • SEQ ID NO:1 is the partial nucleotide sequence of pLoxCat27 encoding the loxP511-Cat-loxP511 cassette.
  • SEQ ID NO:2-3 are oligonucleotide primers used to construct the arcA disruption.
  • SEQ ID NOs:4-5 are oligonucleotide primers used to confirm disruption of arcA.
  • SEQ ID NO:6 is the partial nucleotide sequence of pLoxCat1 encoding the loxP-Cat-loxP cassette.
  • SEQ ID NOs:7-8 are oligonucleotide primers used to construct pR6 KgalP, the template plasmid for trc promoter replacement of the chromosomal galP promoter.
  • SEQ ID NOs:9-10 are oligonucleotide primers used to construct pR6 Kglk, the template plasmid for trc promoter replacement of the chromosomal glk promoter.
  • SEQ ID NO:11 is the nucleotide sequence of the loxP-Cat-loxP-Trc cassette.
  • SEQ ID NOs:12-13 are oligonucleotide primers used to confirm integration of SEQ ID NO:11 for replacement of the chromosomal galP promoter.
  • SEQ ID NOs:14-15 are oligonucleotide primers used to confirm integration of SEQ ID NO:11 for replacement of the chromosomal glk promoter.
  • SEQ ID NOs:16-17 are oligonucleotide primers used to construct the edd disruption.
  • SEQ ID NOs:18-19 are oligonucleotide primers used to confirm disruption of edd.
  • SEQ ID NOs:20 is the nucleotide sequence for the selected trc promoter controlling glk expression.
  • SEQ ID NOs:21 is the partial nucleotide sequence for the standard trc promoter.
  • SEQ ID NOs:22-23 are the oligonucleotide primers used for amplification of gapA.
  • SEQ ID NOs:24-25 are the oligonucleotide primers used to alter the start codon of gapA to GTG.
  • SEQ ID NOs:26-27 are the oligonucleotide primers used to alter the start codon of gapA to TTG.
  • SEQ ID NO:28 is the nucleotide sequence for the short 1.5 GI promoter.
  • SEQ ID NOs:29-30 are oligonucleotide primers used for replacement of the chromosomal gapA promoter with the short 1.5 GI promoter.
  • SEQ ID NO:31 is the nucleotide sequence for the short 1.20 GI promoter.
  • SEQ ID NO:32 is the nucleotide sequence for the short 1.6 GI promoter.
  • SEQ ID NOs:33-34 are oligonucleotide primers used for replacement of the chromosomal gapA promoter with the short 1.20 GI promoter.
  • SEQ ID NO:35 is the oligonucleotide primer with SEQ ID NO 33 that is used for replacement of the chromosomal gapA promoter with the short 1.6 GI promoter.
  • SEQ ID NOs:36-37 are oligonucleotide primers used to construct the mgsA disruption.
  • SEQ ID NOs:38-39 are oligonucleotide primers used to confirm disruption of mgsA.
  • SEQ ID NOs:40-41 are oligonucleotide primers used for replacement of the chromosomal ppc promoter with the short 1.6 GI promoter.
  • SEQ ID NO:42 is an oligonucleotide primer used to confirm replacement of the ppc promoter.
  • SEQ ID NOs:4344 are oligonucleotide primers used for replacement of the chromosomal yciK-btuR promoter with the short 1.6 GI promoter.
  • SEQ ID NOs:45-46 are oligonucleotide primers used to confirm replacement of the yciK-btuR promoter.
  • SEQ ID NOs:47-48 are oligonucleotide primers used for replacement of the chromosomal yqhD promoter with the short 1.6 GI promoter.
  • SEQ ID NO:49 is an oligonucleotide primer used to confirm replacement of the yqhD promoter.
  • SEQ ID NOs:50-51 are oligonucleotide primers used to construct the pta-ackA disruption.
  • SEQ ID NOs:52-53 are oligonucleotide primers used to confirm disruption of pta-ackA.
  • SEQ ID NOs:54-55 are oligonucleotide primers used to construct the ptsHIcrr disruption.
  • SEQ ID NO:56 is an oligonucleotide primer used to confirm disruption of ptsHlcrr.
  • SEQ ID NOs:57-58 are oligonucleotide primers used to construct the aldA disruption.
  • SEQ ID NOs:59-60 are oligonucleotide primers used to confirm disruption of aldA.
  • SEQ ID NOs:61-62 are oligonucleotide primers used to construct the aldB disruption.
  • SEQ ID NOs:63-64 are oligonucleotide primers used to confirm disruption of aldB.
  • SEQ ID NO:65 is the nucleotide sequence for the pSYCO101 plasmid.
  • SEQ ID NO:66 is the nucleotide sequence for the pSYCO103 plasmid.
  • SEQ ID NO:67 is the nucleotide sequence for the pSYCO106 plasmid.
  • SEQ ID NO:68 is the nucleotide sequence for the pSYCO109 plasmid.
  • ATCC refers to the American Type Culture Collection international depository located 10801 University Boulevard., Manassas, Va. 20110-2209 U.S.A.
  • ATCC No.” is the accession number to cultures on deposit with the ATCC.
  • the present invention provides for an improved process for bioconverting a fermentable carbon source directly to 1,3-propanediol using a single microorganism.
  • the method is characterized by improved 1,3-propanediol yield at levels not previously obtained.
  • the disclosed production host strains have been engineered to maximize the metabolic efficiency of the pathway by incorporating various deletion mutations that prevent the diversion of carbon to non-productive compounds. It is contemplated that transformations and mutations can be combined so as to control particular enzyme activities for the enhancement of 1,3-propanediol production. Thus, it is within the scope of the present invention to anticipate modifications of a whole cell catalyst which lead to an increased production of 1,3-propanediol.
  • NADH dehydrogenase II refers to the type II NADH dehydrogenase, a protein that catalyzed the conversion of ubiquinone-8+NADH+H + to ubiquinol-8+NAD + .
  • Typical of NADH dehydrogenase II is EC 1.6.99.3.
  • NADH dehydrogenase II is encoded by ndh in E. coli.
  • the terms “aerobic respiration control protein” and “ArcA” refer to a global regulatory protein.
  • the aerobic respiration control protein is encoded by arcA in E. coli.
  • phosphogluconate dehydratase and “Edd” refer to a protein that catalyzed the conversion of 6-phospho-gluconate to 2-keto-3-deoxy-6-phospho-gluconate+H 2 O.
  • Typical of phosphogluconate dehydratase is EC 4.2.1.12.
  • Phosphogluconate dehydratase is encoded by edd in E. coli.
  • phosphocarrier protein HPr and “PtsH” refer to the phosphocarrier protein encoded by ptsH in E. coli .
  • phosphoenolpyruvate-protein phosphotransferase and “Ptsl” refer to the phosphotransferase, EC 2.7.3.9, encoded by ptsl in E. coli .
  • PTS system and “glucose-specific IIA component”, and “Crr” refer to EC 2.7.1.69, encoded by crr in E. coli .
  • PtsH, Ptsl, and Crr comprise the PTS system.
  • phosphoenolpyruvate-sugar phosphotransferase system refers to the phosphoenolpyruvate-dependent sugar uptake system.
  • methylglyoxal synthase and “MgsA” refer to a protein that catalyzed the conversion of dihydroxy-acetone-phosphate to methyl-glyoxal+phosphate.
  • Typical of methylglyoxal synthase is EC 4.2.3.3.
  • Methylglyoxal synthase is encoded by mgsA in E. coli.
  • aldehyde dehydrogenase A and “AldA” refer to a protein that catalyzed the conversion of H 2 O+NAD + +aldehyde to NADH+alcohol. Typical of aldehyde dehydrogenase A is EC 1.2.1.22. Aldehyde dehydrogenase A is encoded by aldA in E. coli.
  • aldehyde dehydrogenase B and “AldB” refer to a protein that catalyzed the conversion of H 2 O+NAD+ + aldehyde to NADH+alcohol. Typical of aldehyde dehydrogenase B is EC 1.2.1.22. Aldehyde dehydrogenase B is encoded by aldB in E. coli.
  • galactose-proton symporter and “GalP” refer to a protein that catalyses the transport of a sugar and a proton from the periplasm to the cytoplasm. D-glucose is a preferred substrate for GalP.
  • GalP GalP
  • Galactose-proton symporter is encoded by galP in E. coli.
  • glucokinase and “Glk” refer to a protein that catalyses the conversion of D-glucose+ATP to glucose-6-phosphate+ADP. Typical of glucokinase is EC 2.7.1.2. Glucokinase is encoded by glk in E. coli.
  • glycosyl-phosphate dehydrogenase and “GapA” refer to a protein that catalyses the conversion of glyceraldehyde-3-phosphate+phosphate+NAD + to 3-phospho-D-glyceroyl-phosphate+NADH+H + .
  • Typical of glyceraldehyde 3-phosphate dehydrogenase is EC 1.2.1.12.
  • Glyceraldehyde 3-phosphate dehydrogenase is encoded by gapA in E. coli.
  • phosphoenolpyruvate carboxylase and “Ppc” refer to a protein that catalyses the conversion of phosphoenolpyruvate+H 2 O+CO 2 to phosphate+oxaloacetic acid.
  • Typical of phosphoenolpyruvate carboxylase is EC 4.1.1.31.
  • Phosphoenolpyruvate carboxylase is encoded by ppc in E. coli.
  • YciK refers to a putative enzyme encoded by yciK which is translationally coupled to btuR, the gene encoding Cob(I)alamin adenosyltransferase in Escherichia coli.
  • cob(I)alamin adenosyltransferase refers to an enzyme responsible for the transfer of a deoxyadenosyl moiety from ATP to the reduced corrinoid. Typical of cob(I)alamin adenosyltransferase is EC 2.5.1.17.
  • Cob(I)alamin adenosyltransferase is encoded by the gene “btuR” (GenBank M21528) in Escherichia coli , “cobA” (GenBank L08890) in Salmonella typhimurium , and “cobO” (GenBank M62866) in Pseudomonas denitrificans.
  • short 1.20 GI promoter refers to SEQ ID NO:31.
  • short 1.5 GI promoter refers to SEQ ID NO:28.
  • short 1.6 GI promoter and short wild-type promoter are used interchangeably and refer to SEQ ID NO:32.
  • G3PDH glycerol-3-phosphate dehydrogenase
  • DHAP dihydroxyacetone phosphate
  • G3P glycerol-3-phosphate
  • In vivo G3PDH may be NADH; NADPH; or FAD-dependent.
  • NADH-dependent glycerol-3-phosphate dehydrogenase When specifically referring to a cofactor specific glycerol-3-phosphate dehydrogenase, the terms “NADH-dependent glycerol-3-phosphate dehydrogenase”, “NADPH-dependent glycerol-3-phosphate dehydrogenase” and “FAD-dependent glycerol-3-phosphate dehydrogenase” will be used.
  • NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenases are able to use NADH and NADPH interchangeably (for example by the gene encoded by gpsA)
  • NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenase will be used interchangeably.
  • the NADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by several genes including GPD1 (GenBank Z74071 ⁇ 2), or GPD2 (GenBank Z35169 ⁇ 1), or GPD3 (GenBank G984182), or DAR1 (GenBank Z74071 ⁇ 2).
  • GPD1 GenBank Z74071 ⁇ 2
  • GPD2 GeneBank Z35169 ⁇ 1
  • GPD3 GeneBank G984182
  • DAR1 GeneBank Z74071 ⁇ 2
  • the NADPH-dependent enzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and L45246).
  • the FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (GenBank Z47047 ⁇ 23), or glpD (GenBank G147838), or gipABC (GenBank M20938) (see WO 9928480 and references therein, which are herein incorporated by reference).
  • G3P phosphatase refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol-3-phosphate and water to glycerol and inorganic phosphate.
  • G3P phosphatase is encoded, for example, by GPP1 (GenBank Z47047 ⁇ 125), or GPP2 (GenBank U18813x11) (see WO 9928480 and references therein, which are herein incorporated by reference).
  • glycol kinase refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP.
  • the high-energy phosphate donor ATP may be replaced by physiological substitutes (e.g., phosphoenolpyruvate).
  • Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583 ⁇ 19) and glpK (GenBank L 19201) (see WO 9928480 and references therein, which are herein incorporated by reference).
  • glycosyrene dehydrogenase refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C. 1.1.1.72).
  • a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone is also referred to as a “dihydroxyacetone reductase”.
  • Glycerol dehydrogenase may be dependent upon NADH (E.C. 1.1.1.6), NADPH (E.C. 1.1.1.72), or other cofactors (e.g., E.C. 1.1.99.22).
  • a NADH-dependent glycerol dehydrogenase is encoded, for example, by gidA (GenBank U00006) (see WO 9928480 and references therein, which are herein incorporated by reference).
  • dehydratase enzyme or “dehydratase” will refer to any enzyme activity that catalyzes the conversion of a glycerol molecule to the product 3-hydroxypropionaldehyde.
  • the dehydratase enzymes include a glycerol dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28) having preferred substrates of glycerol and 1,2-propanediol, respectively.
  • dehydratase enzymes have been identified in Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium , and Klebsiella oxytoca .
  • the dehydratase is composed of three subunits: the large or “ ” subunit, the medium or “ ⁇ ” subunit, and the small or “ ⁇ ” subunit. Due to the wide variation in gene nomenclature used in the literature, a comparative chart is given in Table 1 to facilitate identification. The genes are also described in, for example, Daniel et al. ( FEMS Microbiol. Rev. 22, 553 (1999)) and Toraya and Mori ( J. Biol. Chem.
  • genes encoding the large or “ ⁇ ” subunit of glycerol dehydratase include dhaB1, gldA and dhaB; genes encoding the medium or “ ⁇ ” subunit include dhaB2, gldB, and dhaC; genes encoding the small or “ ⁇ ” subunit include dhaB3, gldC, and dhaE.
  • genes encoding the large or “ ⁇ ” subunit of diol dehydratase include pduC and pddA; genes encoding the medium or “ ⁇ ” subunit include pduD and pddB; genes encoding the small or “ ⁇ ” subunit include pduE and pddC.
  • GENE FUNCTION 1,3-PD ORGANISM regulatory unknown reactivation dehydrogenase unknown (GenBank base base base base base Reference) gene pairs gene pairs gene pairs gene pairs gene pairs gene pairs gene pairs gene pairs Geme pairs gene pairs gene pairs K.
  • pneumoniae dhaR 2209-4134 orfW 4112-4642 OrfX 4643-4996 dhaT 5017-6108 orfY 6202-6630 (SEQ ID NO: 1)
  • K. pneumoniae orf2c 7116-7646 orf2b 6762-7115 dhaT 5578-6741 orf2a 5125-5556 (U30903)
  • K. pneumoniae GdrB (U60992)
  • OrfX 6180-6533 dhaT 6550-7713 orfY 7736-8164 (U09771)
  • pneumoniae dhaB1 7044-8711 dhaB2 8724-9308 dhaB3 9311-9736 orfZ 9749-11572 SEQ ID NO:1
  • pasteurianum dhaB 84-1748 dhaC 1779-2318 DhaE 2333-2773 orfZ 2790-4598 (AF051373)
  • C. pasteurianum (AF006034)
  • PduE 5921-6442 pduG 6452-8284 (AF026270)
  • K. oxytoca ddrA 241-2073 AF017781
  • K. oxytoca pddA 121-1785 pddB 1796-2470 PddC 2485-3006 (AF051373)
  • Glycerol and diol dehydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)).
  • dehydratase reactivation factor refers to those proteins responsible for reactivating the dehydratase activity.
  • dehydratase reactivating activity refers to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo.
  • Two proteins have been identified as being involved as the dehydratase reactivation factor (see WO 9821341 (US 6013494) and references therein, which are herein incorporated by reference; Daniel et al., supra; Toraya and Mori, J. Biol. Chem.
  • genes encoding one of the proteins include orfZ, dhaB4, gdrA, pduG and ddrA. Also referring to Table 1, genes encoding the second of the two proteins include orfX, orf2b, gdrB, pduH and ddrB.
  • 1,3-propanediol oxidoreductase refers to the polypeptide(s) responsible for an enzyme activity that is capable of catalyzing the interconversion of 3-HPA and 1,3-propanediol provided the gene(s) encoding such activity is found to be physically or transcriptionally linked to a dehydratase enzyme in its natural (i.e., wild type) setting; for example, the gene is found within a dha regulon as is the case with dhaT from Klebsiella pneumoniae.
  • genes encoding a 1,3-propanediol oxidoreductase include dhaT from Klebsiella pneumoniae, Citrobacter freundii , and Clostridium pasteurianum .
  • Each of these genes encode a polypeptide belonging to the family of type III alcohol dehydrogenases, exhibits a conserved iron-binding motif, and has a preference for the NAD + /NADH linked interconversion of 3-HPA and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050 (1987); Daniel et al., J. Bacteriol. 177, 2151 (1995); and Leurs et al., FEMS Microbiol.
  • dha regulon refers to a set of associated genes or open reading frames encoding various biological activities, including but not limited to a dehydratase activity, a reactivation activity, and a 1,3-propanediol oxidoreductase.
  • a dha regulon comprises the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ as described herein.
  • non-specific catalytic activity refers to the polypeptide(s) responsible for an enzyme activity that is sufficient to catalyze the interconversion of 3-HPA and 1,3-propanediol and specifically excludes 1,3-propanediol oxidoreductase(s).
  • these enzymes are alcohol dehydrogenases.
  • Such enzymes may utilize cofactors other than NAD + /NADH, including but not limited to flavins such as FAD or FMN.
  • a gene for a non-specific alcohol dehydrogenase (yqhD) is found, for example, to be endogenously encoded and functionally expressed within E. coli K 12 strains.
  • function refers to the catalytic activity of an enzyme in altering the energy required to perform a specific chemical reaction. It is understood that such an activity may apply to a reaction in equilibrium where the production of either product or substrate may be accomplished under suitable conditions.
  • polypeptide and “protein” are used interchangeably.
  • carbon substrate and “carbon source” refer to a carbon source capable of being metabolized by host microorganisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
  • host cell or “host microorganism” refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product.
  • foreign gene refers to genetic material native to one organism that has been placed within a host microorganism by various means.
  • the gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene.
  • transformation and “transfection” refer to the acquisition of new genes in a cell after the incorporation of nucleic acid.
  • the acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences.
  • transformant refers to the product of a transformation.
  • geometrically altered refers to the process of changing hereditary material by transformation or mutation.
  • the terms “recombinant microorganism” and “transformed host” refer to any microorganism having been transformed with heterologous or foreign genes or extra copies of homologous genes.
  • the recombinant microorganisms of the present invention express foreign genes encoding glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2 and dhaB3), dehydratase reactivation factor (orfZ and orfX), and optionally 1,3-propanediol oxidoreductase (dhaT) for the production of 1,3-propanediol from suitable carbon substrates.
  • GPD1 glycerol-3-phosphate dehydrogenase
  • GPP2 glycerol-3-phosphatase
  • dhaB1, dhaB2 and dhaB3 glycerol
  • a preferred embodiment is an E. coli transformed with these genes but lacking a functional dhaT.
  • a host microorganism, other than E. coli may also be transformed to contain the disclosed genes and the gene for the non-specific catalytic activity for the interconversion of 3-HPA and 1,3-propanediol, specifically excluding 1,3-propanediol oxidoreductase(s) (dhaT).
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding) and following (3′ non-coding) the coding region.
  • regulatory sequences preceding 5′ non-coding
  • 3′ non-coding 3′ non-coding
  • wild-type refer to a gene as found in nature with its own regulatory sequences.
  • encoding and “coding” refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. It is understood that the process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences.
  • isolated refers to a protein or DNA sequence that is removed from at least one component with which it is naturally associated.
  • An “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • substantially similar refers to nucleic acid molecules wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid molecules wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid molecule to mediate alteration of gene expression by antisense or co-suppression technology.
  • “Substantially similar” also refers to modifications of the nucleic acid molecules of the instant invention (such as deletion or insertion of one or more nucleotide bases) that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule.
  • the invention encompasses more than the specific exemplary sequences.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine).
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid
  • one positively charged residue for another such as lysine for arginine
  • nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
  • substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1 ⁇ SSC, 0.1% SDS, 65° C. and washed with 2 ⁇ SSC, 0.1% SDS followed by 0.1 ⁇ SSC, 0.1% SDS), with the sequences exemplified herein.
  • Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.
  • a nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength.
  • Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratorv Manual , Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization.
  • low stringency hybridization conditions corresponding to a Tm of 55°
  • 5 ⁇ SSC 0.1% SDS, 0.25% milk, and no formamide
  • 30% formamide 5 ⁇ SSC, 0.5% SDS
  • Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5 ⁇ or 6 ⁇ SSC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
  • RNA:RNA, DNA:RNA, DNA:DNA For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).
  • the length for a hybridizable nucleic acid is at least about 10 nucleotides.
  • a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides.
  • the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
  • a “substantial portion” refers to an amino acid or nucleotide sequence which comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also www.ncbi.nlm.nih.gov/BLAST/).
  • BLAST Basic Local Alignment Search Tool
  • a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid molecule comprising the primers.
  • a “substantial portion” of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid molecule comprising the sequence.
  • the instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular proteins.
  • the skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for the purpose known to those skilled in the art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • nucleotide bases that are capable to hybridizing to one another.
  • adenosine is complementary to thymine
  • cytosine is complementary to guanine.
  • the instant invention also includes isolated nucleic acid molecules that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
  • identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology , Lesk, A. M., Ed.; Oxford University Press: New York, 1988 ; Biocomputing: Informatics and Genome Projects ; Smith, D.
  • Methods to determine identity and similarity are codified in publicly available computer programs.
  • the BLASTX program is publicly available from NCBI and other sources ( BLAST Manual , Altschul et al., Natl. Cent.
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • homologous refers to a protein or polypeptide native or naturally occurring in a given host cell.
  • the invention includes microorganisms producing homologous proteins via recombinant DNA technology.
  • percent homology refers to the extent of amino acid sequence identity between ppolypeptides. When a first amino acid sequence is identical to a ssecond amino acid sequence, then the first and second amino acid sequences exhibit 100% homology.
  • the homology between any two polypeptides is a direct function of the total number of matching amino acids at a given position in either sequence, e.g., if half of the total number of amino acids in either of the two sequences are the same then the two sequences are said to exhibit 50% homology.
  • Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide.
  • the skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • expression refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
  • Plasmid refers to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
  • Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.
  • Expression cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
  • Recombinant organisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a carbon substrate to 1,3-propanediol may be constructed using techniques well known in the art.
  • Genes encoding glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2, and dhaB3), dehydratase reactivation factor (orfZ and orfX) and 1,3-propanediol oxidoreductase (dhaT) were isolated from a native host such as Klebsiella or Saccharomyces and used to transform host strains such as E. coli DH5 ⁇ , ECL707, M200, or KLP23.
  • PCR polymerase chain reaction
  • cosmid libraries may be created where large segments of genomic DNA (3545 kb) may be packaged into vectors and used to transform appropriate hosts.
  • Cosmid vectors are unique in being able to accommodate large quantities of DNA.
  • cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the foreign DNA.
  • these vectors will also contain an origin of replication such as ColE1 and drug resistance markers such as a gene resistant to ampicillin or neomycin.
  • cosmid vectors containing the linearized foreign DNA are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process the cos sites are cleaved and the foreign DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli . Once injected into the cell, the foreign DNA circularizes under the influence of the cos sticky ends. In this manner large segments of foreign DNA can be introduced and expressed in recombinant host cells.
  • Cosmid vectors and cosmid transformation methods were used within the context of the present invention to clone large segments of genomic DNA from bacterial genera known to possess genes capable of processing glycerol to 1,3-propanediol.
  • genomic DNA from K. pneumoniae was isolated by methods well known in the art and digested with the restriction enzyme Sau3A for insertion into a cosmid vector Supercos 1 and packaged using GigapackII packaging extracts.
  • E. coli XL1-Blue MR cells were transformed with the cosmid DNA. Transformants were screened for the ability to convert glycerol to 1,3-propanediol by growing the cells in the presence of glycerol and analyzing the media for 1,3-propanediol formation.
  • the instant invention utilizes the isolated genes from within a Klebsiella cosmid, alternate sources of dehydratase genes and dehydratase reactivation factor genes include, but are not limited to, Citrobacter, Clostridia and Salmonella (see Table 1).
  • the present invention provides genes suitable for the expression of G3PDH and G3P phosphatase activities in a host cell.
  • GPD1 has been isolated from Saccharomyces (Wang et al., supra).
  • G3PDH activity has also been isolated from Saccharomyces encoded by GPD2 (Eriksson et al., Mol. Microbiol. 17, 95 (1995)).
  • any gene encoding a polypeptide responsible for NADH-dependent G3PDH activity is suitable wherein that activity is capable of catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P).
  • DHAP dihydroxyacetone phosphate
  • G3P glycerol-3-phosphate
  • any gene encoding the amino acid sequence of NADH-dependent G3PDH's corresponding to the genes DAR1, GPD1, GPD2, GPD3, and gpsA will be functional in the present invention wherein that amino acid sequence may encompass amino acid substitutions, deletions or additions that do not alter the function of the enzyme.
  • G3PDH genes encoding G3PDH isolated from other sources will also be suitable for use in the present invention.
  • Genes encoding G3P phosphatase are known.
  • GPP2 has been isolated from Saccharomyces cerevisiae (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)).
  • any gene encoding a G3P phosphatase activity is suitable for use in the method wherein that activity is capable of catalyzing the conversion of glycerol-3-phosphate plus H 2 O to glycerol plus inorganic phosphate.
  • any gene encoding the amino acid sequence of G3P phosphatase corresponding to the genes GPP2 and GPP1 will be functional in the present invention including any amino acid sequence that encompasses amino acid substitutions, deletions or additions that do not alter the function of the G3P phosphatase enzyme.
  • genes encoding G3P phosphatase isolated from other sources will also be suitable for use in the present invention.
  • Suitable host cells for the recombinant production of 1,3-propanediol may be either prokaryotic or eukaryotic and will be limited only by the host cell ability to express the active enzymes for the 1,3-propanediol pathway.
  • Suitable host cells will be microorganisms such as Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas.
  • Preferred in the present invention are Escherichia coli, Escherichia blattae , Klebsiella, Citrobacter, and Aerobacter. Most preferred is E. coli (KLP23 (WO 2001012833 A2), RJ8.n (ATCC PTA4216), E. coli : FMP′::Km (ATCC PTA4732), MG 1655 (ATCC 700926)).
  • the present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor into a suitable host cell.
  • Suitable vectors will be those which are compatible with the microorganism employed.
  • Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and using such vectors are known to those in the art (Sambrook et al., supra).
  • Initiation control regions, or promoters, which are useful to drive expression of the G3PDH and G3P phosphatase genes (DAR1 and GPP2, respectively) in the desired host cell are numerous and familiar to those skilled in the art.
  • Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, ⁇ P L , ⁇ P R , T7, tac, and trc (useful for expression in E. coli ).
  • Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
  • DNA encoding the enzymes are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.
  • Particularly useful in the present invention are the vectors pSYCO101, pSYCO103, pSYCO106, and pSYCO109.
  • the essential elements are derived from the dha regulon isolated from Klebsiella pneumoniae and from Saccharomyces cerevlsiae .
  • Each contains the open reading frames dhaB1, dhaB2, dhaB3, dhaX, orfX, DAR1, and GPP2 arranged in three separate operons, nucleotide sequences of which are given in SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively.
  • the differences between the vectors are illustrated in the chart below [the prefix “p-” indicates a promoter; the open reading frames contained within each “( )” represent the composition of an operon]:
  • cassettes are used to transform appropriate host cells.
  • Introduction of the cassette containing the genes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor into the host cell may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, electroporation), or by transfection using a recombinant phage virus (Sambrook et al., supra).
  • cassettes were used to transform the E. coli as fully described in the GENERAL METHODS and EXAMPLES.
  • the present method will be able to make use of cells having single or multiple mutations specifically designed to enhance the production of 1,3-propanediol.
  • Cells that normally divert a carbon feed stock into non-productive pathways, or that exhibit significant catabolite repression could be mutated to avoid these phenotypic deficiencies.
  • many wild-type cells are subject to catabolite repression from glucose and by-products in the media and it is contemplated that mutant strains of these wild-type organisms, capable of 1,3-propanediol production that are resistant to glucose repression, would be particularly useful in the present invention.
  • UV radiation in this wavelength principally causes changes within nucleic acid sequence from guanidine and cytosine to adenine and thymidine. Since all cells have DNA repair mechanisms that would repair most UV induced mutations, agents such as caffeine and other inhibitors may be added to interrupt the repair process and maximize the number of effective mutations. Long wave UV mutations using light in the 300 nm to 400 nm range are also possible but are generally not as effective as the short wave UV light unless used in conjunction with various activators such as psoralen dyes that interact with the DNA.
  • Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect nonreplicating DNA such as HNO 2 and NH 2 OH, as well as agents that affect replicating DNA such as acridine dyes, notable for causing frameshift mutations.
  • Specific methods for creating mutants using radiation or chemical agents are well documented in the art. See, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology , Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA., or Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992), herein incorporated by reference.
  • mutants having the desired phenotype may be selected by a variety of methods. Random screening is most common where the mutagenized cells are selected for the ability to produce the desired product or intermediate. Alternatively, selective isolation of mutants can be performed by growing a mutagenized population on selective media where only resistant colonies can develop. Methods of mutant selection are highly developed and well known in the art of industrial microbiology. See for example Brock, Supra; DeMancilha et al., Food Chem. 14, 313 (1984).
  • genes involved in converting carbon substrate to 1,3-propanediol may be up-regulated or down-regulated by a variety of methods which are known to those skilled in the art. It is well understood that up-regulation or down-regulation of a gene refers to an alteration in the activity of the protein encoded by that gene relative to a control level of activity, for example, by the activity of the protein encoded by the corresponding (or non-altered) wild-type gene.
  • Specific genes involved in an enzyme pathway may be up-regulated to increase the activity of their encoded function(s). For example, additional copies of selected genes may be introduced into the host cell on multicopy plasmids such as pBR322. Such genes may also be integrated into the chromosome with appropriate regulatory sequences that result in increased activity of their encoded functions.
  • the target genes may be modified so as to be under the control of non-native promoters or altered native promoters. Endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution.
  • Down-regulation can occur by deletion, insertion, or alteration of coding regions and/or regulatory (promoter) regions.
  • Specific down regulations may be obtained by random mutation followed by screening or selection, or, where the gene sequence is known, by direct intervention by molecular biology methods known to those skilled in the art.
  • a particularly useful, but not exclusive, method to effect down-regulation is to alter promoter strength.
  • Disruptions of genes may occur, for example, by 1) deleting coding regions and/or regulatory (promoter) regions, 2) inserting exogenous nucleic acid sequences into coding regions and/regulatory (promoter) regions, and 3) altering coding regions and/or regulatory (promoter) regions (for example, by making DNA base pair changes). Such changes would either prevent expression of the protein of interest or result in the expression of a protein that is non-functional.
  • Specific disruptions may be obtained by random mutation followed by screening or selection, or, in cases where the gene sequences in known, specific disruptions may be obtained by direct intervention using molecular biology methods know to those skilled in the art.
  • a particularly useful method is the deletion of significant amounts of coding regions and/or regulatory (promoter) regions.
  • Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis.
  • DHA dihydroxyacetone
  • G3P glycerol 3-phosphate
  • the hydrolysis step can be catalyzed by any number of cellular phosphatases, which are known to be non-specific with respect to their substrates, or the activity can be introduced into the host by recombination.
  • the reduction step can be catalyzed by a NAD + (or NADP + ) linked host enzyme or the activity can be introduced into the host by recombination.
  • NAD + or NADP +
  • the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) that catalyzes the reversible reaction of Equation 3.
  • Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxy-propionaldehye (3-HPA) as has been described in detail above.
  • the intermediate 3-HPA is produced from glycerol, Equation 1, by a dehydratase enzyme that can be encoded by the host or can be introduced into the host by recombination.
  • This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or any other enzyme able to catalyze this transformation.
  • Glycerol dehydratase, but not diol dehydratase is encoded by the dha regulon.
  • 1,3-Propanediol is produced from 3-HPA, Equation 2, by a NAD + - (or NADP + ) linked host enzyme or the activity can be introduced into the host by recombination.
  • This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.
  • Mutations and transformations that affect carbon channeling A variety of mutant microorganisms comprising variations in the 1,3-propanediol production pathway will be useful in the present invention. Mutations which block alternate pathways for intermediates of the 1,3-propanediol production pathway would also be useful to the present invention. For example, the elimination of glycerol kinase prevents glycerol, formed from G3P by the action of G3P phosphatase, from being re-converted to G3P at the expense of ATP.
  • glycerol dehydrogenase for example, gidA
  • gidA glycerol dehydrogenase
  • Mutations can be directed toward a structural gene so as to impair or improve the activity of an enzymatic activity or can be directed toward a regulatory gene, including promoter regions and ribosome binding sites, so as to modulate the expression level of an enzymatic activity.
  • transformations and mutations can be combined so as to control particular enzyme activities for the enhancement of 1,3-propanediol production.
  • the present invention utilizes a preferred pathway for the production of 1,3-propanediol from a sugar substrate where the carbon flow moves from glucose to DHAP, G3P, Glycerol, 3-HPA, and finally to 1,3-propanediol.
  • the present production strains have been engineered to maximize the metabolic efficiency of the pathway by incorporating various deletion mutations that prevent the diversion of carbon to non-productive compounds.
  • Glycerol may be diverted from conversion to 3HPA by transformation to either DHA or G3P via glycerol dehydrogenase or glycerol kinase as discussed above. Accordingly, the present production strains contain deletion mutations in the gidA and glpk genes.
  • DHAP may be diverted to 3-PG by triosephosphate isomerase, thus the present production microorganism also contains a deletion mutation in this gene.
  • the present method additionally incorporates a dehydratase enzyme for the conversion of glycerol to 3HPA, which functions in concert with the reactivation factor, encoded by orfX and orfZ of the dha regulon.
  • conversion of the 3HPA to 1,3-propanediol is typically accomplished via a 1,3-propanediol oxidoreductase
  • the present method utilizes a non-specific catalytic activity that produces greater titers and yields of the final product, 1,3-propanediol. In such a process, titers of 1,3-propanediol of at least 10 g/L are achieved, where titers of 200 g/L are expected.
  • an improved process for 1,3-propanediol production may utilize glycerol or dihydroxyacetone as a substrate where the pathway comprises only the last three substrates, glycerol ⁇ 3HPA ⁇ 1,3-propanediol.
  • the oxidoreductase is again eliminated in favor of the non-specific catalytic activity (expected to be an alcohol dehydrogenase), however the need for deletion mutations are nullified by the energy considerations of adding glycerol to the culture.
  • titers of 1,3-propanediol of at least 71 g/L are achieved where titers of 200 g/L are expected.
  • mutants of wild-type microorganisms that have been modified by the deletion or mutation of the dhaT activity to create improved 1,3-propandiol producers.
  • microorganisms which naturally contain all the elements of the dha regulon, may be manipulated so as to inactivate the dhaT gene encoding the 1,3-propanediol oxidoreductase activity. These microorganisms will be expected to produce higher yields and titers of 1,3-propanediol, mediated by the presence of an endogenous catalytic activity, expected to be an alcohol dehydrogenase. Examples of such microorganisms include but are not limited to Klebsiella sp., Citrobacter sp., and Clostridium sp.
  • Fermentation media in the present invention must contain suitable carbon substrates.
  • suitable substrates may include but are not limited to monosaccharides such as glucose and fructose and oligosaccharides such as lactose or sucrose.
  • the preferred carbon substrate is glucose
  • fermentation media In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 1,3-propanediol production. Particular attention is given to Co(II) salts and/or vitamin B 12 or precursors thereof.
  • Adenosyl-cobalamin (coenzyme B 12 ) is an essential cofactor for dehydratase activity. Synthesis of coenzyme B 12 is found in prokaryotes, some of which are able to synthesize the compound de novo, for example, Escherichia blattae , Klebsiella species, Citrobacter species, and Clostridium species, while others can perform partial reactions. E. coli , for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to corrinoid and can introduce the 5′-deoxyadenosyl group. Thus, it is known in the art that a coenzyme B 12 precursor, such as vitamin B 12 , need be provided in E. coli fermentations.
  • Vitamin B 12 additions to E. coli fermentations may be added continuously, at a constant rate or staged as to coincide with the generation of cell mass, or may be added in single or multiple bolus additions.
  • Preferred ratios of vitamin B 12 (mg) fed to cell mass (OD550) are from 0.06 to 0.60. Most preferred ratios of vitamin B 12 (mg) fed to cell mass (OD550) are from 0.12 to 0.48.
  • vitamin B 12 is added to the transformed E. coli of the present invention it is contemplated that other microorganisms, capable of de novo B 12 biosynthesis will also be suitable production cells and the addition of B 12 to these microorganisms will be unnecessary.
  • Preferred growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth.
  • LB Luria Bertani
  • SD Sabouraud Dextrose
  • YM Yeast medium
  • Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by someone skilled in the art of microbiology or fermentation science.
  • agents known to modulate catabolite repression directly or indirectly e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the reaction media.
  • agents known to modulate enzymatic activities e.g., methyl viologen
  • agents known to modulate enzymatic activities e.g., methyl viologen
  • Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.
  • Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism.
  • Fed-batch fermentations may be performed with carbon feed, for example, glucose, limited or excess.
  • the present process employs a batch method of fermentation.
  • Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the media is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur adding nothing to the system.
  • “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration.
  • the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped.
  • cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die.
  • Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
  • a variation on the standard batch system is the Fed-Batch system.
  • Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses.
  • Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO 2 .
  • Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, supra.
  • Continuous fermentation is an open system where a defined fermentation media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
  • Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration.
  • one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate.
  • a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant.
  • Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the fermentation.
  • the present invention may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 1,3-propanediol production.
  • propanediols can be obtained from cell media by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. Pat. No. 5,356,812).
  • organic solvent for this process is cyclohexane (U.S. Pat. No. 5,008,473).
  • 1,3-Propanediol may be identified directly by submitting the media to high pressure liquid chromatography (HPLC) analysis.
  • HPLC high pressure liquid chromatography
  • Preferred in the present invention is a method where fermentation media is analyzed on an analytical ion exchange column using a mobile phase of 0.01 N sulfuric acid in an isocratic fashion.
  • loxP/Cre recombinase system from P1 phage and the FRT/Flp recombinase system from yeast provide a mechanism to remove the marker.
  • the loxP and FRT sites are recognition sites for the Cre and Flp recombinases. Cre and Flp are site specific recombinases, which excise the intervening DNA from the directly repeated recognition sites.
  • Strains integrated with a loxP cassette are transformed with pJW168 that encodes Cre recombinase (Palmeros et al, supra).
  • Strains containing a FRT cassette are transformed with pCP20 that encodes Fip recombinase (Datsenko and Wanner, supra). After removal of the integrated marker, the recombinase plasmids are cured from the strain.
  • P1 virtransduction were performed as previously described [Miller, J. H., A short course in bacterial genetics. A laboratory manual and handbook for Escherchia coli and related bacteria (1992), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.].
  • Glucokinase (Glk) activity was assayed by following the conversion of glucose to glucose-6-phosphate spectrophotometrically at 340 nm by coupling the glucokinase reaction with that of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49).
  • the assay contained 0.5 mM NADP, 5 mM ATP, 5 mM MgCl, and 2 units of glucose-6-phosphate dehydrogenase in 100 mM phosphate buffer, pH 7.2.
  • Alternative assays may be found in T. E. Barman, Enzyme Handbook (1985), Springer-Verlag, Berlin.
  • Assay for Glyceraldehyde-3-phosphate dehydrogenase activity was measured in cell-free extracts by the appearance of NADH. An ultracentrifuged (50,000 ⁇ g, 1 h, 4° C.) cell-free supernatant was partially purified using an ion exchange column prior to assay. The assay contained 0.2 mM glyceraldehyde 3-phosphate, 2.5 mM NAD + , 2 mM EDTA, 5 mM cysteamine, 20 mM potassium phosphate and 40 mM triethanolamine at pH 8.9. Alternative assays may be found in T. E. Barman, supra.
  • Phosphoenolpyruvate carboxylase (Ppc) activity was measured in cell-free extracts by a coupled assay (Flores and Gancedo, FEBS Lett. 412, 531-534 (1997)). This method involved incubating at room temperature a ultracentifuged (50,000 ⁇ g, 1 h, 4° C.) cell-free extract sample in a cuvette that contained 0.22 mM NADH, 1.1 mM phosphoenolpyruvate (PEP), 0.25 mM acetyl-CoA, and 6 U of malate dehydrogenase (MDH) in 0.1 M Tris/HCl buffer, pH 8.5, with 11 mM sodium bicarbonate and 11 mM magnesium sulfate, in a total volume of 1.0 mL.
  • PEP 1.1 mM phosphoenolpyruvate
  • MDH malate dehydrogenase
  • a background rate of the reaction of enzyme and NADH was first determined at 340 nm in the absence of PEP.
  • the second substrate, PEP was subsequently added and the absorbance change over time was further monitored.
  • Ppc activity was defined by subtracting the background rate from the gross rate.
  • Alternative assays may be found in T. E. Barman, supra.
  • Dehydratase activity in cell-free extracts was determined using either glycerol or 1,2-propanediol as substrate.
  • cell-free extracts were prepared by cell disruption using a french press followed by centrifugation of the cellular debris.
  • the assay based on the reaction of aldehydes with methylbenzo-2-thiazolone hydrazone, has been described by Forage and Foster ( Biochim. Biophys. Acta 569, 249 (1979)).
  • 1,3-propanediol oxidoreductase sometimes referred to as 1,3-propanediol dehydrogenase
  • 1,3-propanediol dehydrogenase The activity of 1,3-propanediol oxidoreductase, sometimes referred to as 1,3-propanediol dehydrogenase, was determined for cell-free extracts in solution or in slab gels using 1,3-propanediol and NAD + as substrates has been described (Johnson and Lin, J. Bacteriol. 169, 2050 (1987)).
  • the conversion of 3-HPA and NADH to 1,3-propanediol and NAD + was determined by the disappearance of NADH.
  • the slab gel assay has the potential advantage of separating the activity of 1,3-propanediol oxidoreductase (dhaT) from that of non-specific alcohol dehydrogenases by virtue of size separation.
  • dhaT 1,3-propanediol oxidoreductases
  • Lactobacillus brevis and Lactobacillus buchneri contain dehydratase associated 1,3-propanediol oxidoreductases with properties similar to those of known 1,3-propanediol oxidoreductases (dhaT).
  • the assay for enzyme activity was performed by incubating the extract with an organic phosphate substrate in a bis-Tris or MES and magnesium buffer, pH 6.5.
  • the substrate used was either I- ⁇ -glycerol phosphate, or d,l- ⁇ -glycerol phosphate.
  • the final concentrations of the reagents in the assay are: buffer (20 mM, bis-Tris or 50 mM MES); MgCl 2 (10 mM); and substrate (20 mM). If the total protein in the sample was low and no visible precipitation occurs with an acid quench, the sample was conveniently assayed in the cuvette.
  • This method involved incubating an enzyme sample in a cuvette that contained 20 mM substrate (50 ⁇ L, 200 mM), 50 mM MES, 10 mM MgCl 2 , pH 6.5 buffer. The final phosphatase assay volume was 0.5 mL.
  • the enzymatic reaction was quenched by the addition of the acid molybdate reagent (0.4 mL).
  • NADH-linked glycerol dehydrogenase activity (gidA) in cell-free extracts from E. coli strains was determined after protein separation by non-denaturing polyacrylamide gel electrophoresis.
  • the conversion of glycerol plus NAD + to dihydroxyacetone plus NADH was coupled with the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to a deeply colored formazan, using phenazine methosulfate (PMS) as mediator (Tang et al., J. Bacteriol. 140, 182 (1997)).
  • Electrophoresis was performed in duplicate by standard procedures using native gels (8-16% TG, 1.5 mm, 15 lane gels from Novex, San Diego, Calif.). Residual glycerol was removed from the gels by washing 3 ⁇ with 50 mM Tris or potassium carbonate buffer, pH 9 for 10 min. The duplicate gels were developed, with and without glycerol (approximately 0.16 M final concentration), in 15 mL of assay solution containing 50 mM Tris or potassium carbonate, pH 9, 60 mg ammonium sulfate, 75 mg NAD+, 1.5 mg MTT, and 0.5 mg PMS.
  • HPLC analysis of fermentation products The conversion of glucose to 1,3-propanediol was monitored by HPLC. Analyses were performed using standard chromatography. One suitable method utilized a Waters Alliance HPLC system using R1 detection. Samples were injected onto a Aminex HPX87H column (7.8 mm ⁇ 300 mm, Biorad, Hercules, Calif.) equipped with a Cation H Refill Cartridge precolumn (4.6 mm ⁇ 30 mm, Biorad, Hercules, Calif.), temperature controlled at 50° C., using 5 mM H 2 SO 4 as mobile phase at a flow rate of 0.4 mL/min. The system was calibrated weekly against standards of known concentration. Typically, the retention times of glucose, glycerol, 1,3-propanediol, and acetic acid were 12.7 min, 19.0 min, 25.2 min, and 21.5 min, respectively.
  • An alternative method for GC/MS involved derivatization of the sample.
  • sample e.g., culture supernatant
  • 30 ⁇ L of concentrated (70% v/v) perchloric acid After mixing, the sample was frozen and lyophilized.
  • a 1:1 mixture of bis(trimethylsilyl)trifluoroacetamide:pyridine (300 ⁇ L) was added to the lyophilized material, mixed vigorously and placed at 65° C. for one h.
  • the sample was clarified of insoluble material by centrifugation.
  • the resulting liquid partitioned into two phases, the upper of which was used for analysis.
  • the sample was chromatographed on a DB-5 column (48 m, 0.25 mm I.D., 0.25 ⁇ m film thickness; from J&W Scientific) and the retention time and mass spectrum of the 1,3-propanediol derivative obtained from culture supernatants were compared to that obtained from authentic standards.
  • the mass spectra of TMS-derivatized 1,3-propanediol contains the characteristic ions of 205, 177, 130 and 115 AMU.
  • TM2 medium is a base recipe to which carbon source (typically glucose, at 20 g/L or 40 g/L), appropriate antibiotics, and other components are added.
  • TM2 medium contains the following components: K 2 HPO 4 (13.6 g/L), KH 2 PO 4 (13.6 g/L), MgSO 4 7H 2 O (2 g/L), citric acid monohydrate (2 g/L), ferric ammonium citrate (0.3 g/L), (NH 4 ) 2 SO 4 (3.2 g/L), yeast extract (5 g/L), solution of trace elements (1 ml). The pH is adjusted to 6.8.
  • the solution of trace elements contains: citric acid H 2 O (4.0 g/L), MnSO 4 .H 2 O (3.0 g/L), NaCl (1.0 g/L), FeSO 4 .7H 2 O (0.10 g/L), COCl 2 .6H 2 O (0.10 g/L), ZnSO 4 .7H 2 O (0.10 g/L), CuSO 4 .5H 2 O (0.010 g/L), H 3 BO 3 (0.010 g/L), and Na 2 MoO 4 .2H 2 O (0.010 g/L).
  • TM3 medium (TM3) is identical to TM2 medium except that it contains 0.5 g/L yeast extract.
  • LB medium contains 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl.
  • LB plates are LB medium+2% agar.
  • 2YT medium contains 10 g/L yeast extract, 16 g/L tryptone, and 10 g/L NaCl.
  • Soy broth with glucose SBG1%
  • Soytone Difco
  • 5 g/L yeast extract 10 g/L NaCl
  • 10 g/L glucose g/L glucose
  • the cultures were incubated at 34° C. and 300 rpm shaking to an OD 550 of approximately 1.0 AU was reached and used to seed the fermenter. In some cases, a seed fermenter was used to provide a larger pre-culture for inoculating a production fermenter. Seed fermenters were generally identical to production fermenters except that vitamin B 12 was not added to the seed tank. Details about the procedures for using seed fermenters are described in the pertinent examples.
  • Seed and production fermenters were prepared with the same medium, containing KH 2 PO 4 (7.5 g/L), MgSO 4 .7H 2 O (2 g/L), citric acid monohydrate (2 g/L), ferric ammonium citrate (0.3 g/L), CaCl 2 ,2H 2 O (0.2 g/L), sulfuric acid (98%; 1.2 mL/L), Mazu DF204 (0.4 mL/L) as antifoam, yeast extract (5 g/L), solution of trace elements (10 ml/L).
  • the solution of trace elements contains: citric acid H 2 O (4.0 g/L), MnSO 4 .H 2 O (3.0 g/L), NaCl (1.0 g/L), FeSO 4 .7H 2 O (0.10 g/L), COCl 2 .6H 2 O (0.10 g/L), ZnSO 4 . 7H 2 O (0.10 g/L), CuSO 4 .5H 2 O (0.010 g/L), H 3 BO 3 (0.010 g/L), and Na 2 MoO 4 .2H 2 O (0.010 g/L).
  • the fermenter volume after inoculation was 6 liters.
  • a 14 L stirred tank fermenter was prepared with the medium described above. The temperature was controlled at 34° C. and aqueous ammonia (20-28 weight %) was used to control pH at 6.8. Backpressure was controlled at 0.5 barg and dO control set at 5%. Except for minor excursions, glucose concentration was maintained between 0 g/L and 25 g/L with a 60-67% (w/w) feed. Vitamin B 12 additions and any other changes to the general procedure described here are noted in the examples.
  • Molar yield as either fraction or %, represents (mol glycerol produced+mol 1,3-propanediol produced)/(mol glucose consumed).
  • Weight yield generally given as %, represents (g 1,3-propanediol produced)/(g glucose consumed).
  • ndh mutation was obtained by interrupting the coding region with a loxP511 cassette.
  • the ndh gene (for reference, see GenBank, Accession # U00096), with upstream and downstream flanking regions, was PCR amplified from E. coli MG1655 and cloned.
  • the ndh cassette was digested with Stu I, cutting roughly in the middle of the gene, and a loxP511-Cat-loxP511 cassette was cloned into this site with the cat gene in the opposite orientation relative to the ndh gene.
  • the loxP511-Cat-/oxP511 cassette was obtained from the plasmid pLoxCat27 [SEQ ID NO:1] by digestion with Spe I and EcoR V, followed by fill-in to generate blunt ends, and gel purification of the 1.1 kb fragment.
  • the loxP511 site is a variant of the loxP site (Palmeros et al., supra).
  • the ndh::Cat cassette was PCR amplified and electroporated into KLP23 competent cells creating strain KLndh81::Cm.
  • the chloramphenicol marker was removed by the Cre recombinase (Palmeros et al., supra) leaving 96 bp of interrupting sequence containing one loxP511 site.
  • This strain was designated KLndh81.
  • an ndh mutation was obtained by interrupting the coding region with a Cat cassette without loxP sites to give strain KLNDH413.
  • a cassette containing ndh flanking sequence and loxP511-Cat-loxP511 from Klndh81::Cm was PCR amplified and cloned into pUni/V5-His TOPO [Invitrogen] to create pAH111.
  • the ndh-loxP511-Cat-loxP511 cassette from pAH111 was integrated into strain RJ8/pKD46. Recombinant strains were selected for chloramphenicol resistance. Successful integration of the cassette into ndh was confirmed by PCR.
  • the chloramphenicol marker was removed by using Cre recombinase (Palmeros et al., supra) creating strain RJ8.n.
  • Strain KLP23 was transformed with plasmids pAH48 and pDT29 or pKP32. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods. Pre-cultures for each fermentation were prepared using frozen vials thawed and grown in 500 mL 2YT with 200 mg/L carbenicillin and 50 mg/L spectinomycin. The full contents of the flask were used to inoculate the fermenter. The fermenter was operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods. The vitamin B 12 strategy for each fermentation is detailed below.
  • vitamin B 12 (0.075 g/L, 500 mL) was fed, starting 3 h after inoculation, at a rate of 16 mL/h.
  • a representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pDT29 is given in Table 2.1.
  • the yield of 1,3-propanediol was 24 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 68 g/L 1,3-propanediol was obtained.
  • a representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pKP32 is given in Table 2.2.
  • Vitamin B 12 (0.150 g/L, 500 mL) was fed, starting 3 h after inoculation, at a rate of 16 mL/h. After 36 h, approximately 2 L of fermentation broth was purged in order to allow for the continued addition of glucose feed.
  • the yield of 1,3-propanediol was 26 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 112 g/L 1,3-propanediol was obtained.
  • RJ8/pAH48pDT29 and RJ8/pAH48/pKP32 pre-cultures were prepared using frozen vials thawed and grown in 500 mL 2YT with 200 mg/L carbenicillin and 50 mg/L spectinomycin. The full contents of the flask were used to inoculate the fermenter. The fermenter was operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods.
  • RJ8/pAH48/pKP32 is identical to RJ8/pAH48/pDT29 except that dhaT is deleted.
  • the vitamin B 12 strategy for each fermentation is detailed below.
  • a representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8/pAH48/pDT29 is given in Table 2A.1.
  • Vitamin B 12 was provided as bolus additions of 2, 16 and 16 mg at 2, 8, and 26 h, respectively.
  • the yield of 1,3-propanediol was 35 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 50.1 g/L 1,3-propanediol was obtained.
  • TABLE 2A.1 Representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E.
  • a representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8/pAH48/pKP32 is given in Table 2A.2.
  • Vitamin B 12 was provided as bolus additions of 48 and 16 mg at approximately 26 and 44 hr, respectively.
  • the yield of 1,3-propanediol was 34 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 129 g/L 1,3-propanediol was obtained.
  • TABLE 2A.2 Representative fermentation summary of the improved conversion of glucose to 1,3-propanediol (1,3-PD) using E.
  • Strain RJ8.n was transformed with plasmids pAH48 and pKP32. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods. A thawed frozen vial of RJ8.n/pAH48/pKP32 was transferred to 500 mL LB with 200 mg/L carbenicillin and 50 mg/L spectinomycin to prepare the pre-culture. The culture was transferred to a seed fermenter and grown for 16 h before 1 L of the culture was transferred to the production fermenter. At that time, the OD550 had reached over 50 AU and 30 g/L glycerol had accumulated in the broth. Both the seed and production fermenters were operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods.
  • Vitamin B 12 was added to the production tank in 16 mg boluses at 12 h, 17.3 h, 22.8 h, and 27.5 h.
  • the final titer was 112.7 g/L 3G and the mass yield was 31.6%.
  • An arcA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 0.6 kb of the coding region with the FRT-CmR-FRT cassette of pKD3.
  • a replacement cassette was amplified with the primer pair SEQ ID NO:2 and SEQ ID NO:3 using pKD3 as the template.
  • the primer SEQ ID NO:2 contains 41 bp of homology to the 5′ end of arcA and 20 bp of homology to pKD3.
  • Primer SEQ ID NO:3 contains 42 bp of homology to the 3′ end of arcA and 20 bp of homology to pKD3.
  • the PCR product was gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of chloramphenicol. The deletion of the arcA gene was confirmed by PCR, using the primer pair SEQ ID NO:4 and SEQ ID NO:5. The wild-type strain gives a 0.7 kb PCR product while the recombinant strain gives a characteristic 1.1 kb PCR product. The strain has been designated MG1655 AarcA::Cm. A P1 lysate was prepared and used to move the mutation into the strain KLndh81 to form KLndh81 ⁇ arcA::Cm.
  • the KLndh81 ⁇ arcA::Cm strain and KLndh81 control strain were electrotransformed with plasmid pSYCO101.
  • One colony of each strain was incubated 10 h in LB medium with 50 mg/L spectinomycin.
  • a 200 ⁇ L volume of these cultures was then transferred to a 250 mL baffled Erlenmeyer flask containing 10 mL TM2 medium with 40 g/L glucose, 50 mg/L spectinomycin, and 2 mg/L vitamin B 12 .
  • the flasks were incubated at 300 rpm and 34° C. for 40 hrs.
  • PPS ⁇ Phosphotranserase System Minus
  • Linear DNA was obtained from plasmid pTrc99a (Pharmacia) digested with HindIII and NcoI, filled with T4 DNA polymerase, circularized and transformed into E. coli TOP-10 (Invitrogen, Carlsbad, Calif.). Following selection on Luria-agar plates containing 50 mg/L of carbenicillin, the resulting plasmid (pTrcl) was purified and subjected to restriction enzyme analysis to confirm that the DNA region originally present between HindIII and NcoI was absent.
  • the unique BspM1 site in pTrc1 (upstream of the ⁇ 35 region of the trc promoter) was used to insert a cassette containing a chloramphenicol resistance gene (CAT) flanked by loxP sites.
  • Linear DNA was obtained from pTrc1 digested with BspM1, gel-purified using a QIAquick gel extraction kit (QIAGEN), filled in with T4 DNA polymerase, and ligated to a loxP-Cat-loxP cassette.
  • the loxP-Cat-loxP cassette was obtained from the plasmid pLoxCat1, see SEQ ID NO:6, by digestion with Stu1 and BamH1.
  • pLoxCat1 is similar to pLoxCat2 (Palmeros et al, supra].
  • the ligation mixture was transformed into E. coli TOP-10 (Invitrogen) and selection was performed on Luria-agar plates containing 50 mg/L of carbenicillin and 20 mg/L of chloramphenicol. Plasmid was obtained and restriction enzyme analysis performed.
  • a DNA cassette containing the trc promoter and lac operator with an upstream loxP-CAT-loxP cassette was PCR amplified from pTrCm42 using the primer pair SEQ ID NO:7 and SEQ ID NO:8.
  • the primer pair incorporates 40 bp of homology to the galP upstream region to each end of the PCR product.
  • PCR parameters were 95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min, 30 cycles using Taq polymerase (Roche).
  • the product was subcloned into Echo pUni/His5 R6K (Invitrogen) to generate the plasmid pR6 KgalP.
  • a DNA cassette containing the trc promoter and lac operator with an upstream loxP-CAT-loxP cassette was amplified from pTrCm42 by PCR using the primer pair SEQ ID NO:9 and SEQ ID NO:10.
  • the primer pair SEQ ID NO:9 and SEQ ID NO:10 incorporates 39 (with a one base deletion) and 40 bp of homology, respectively, to the glk upstream region to each end of the PCR product.
  • PCR parameters were 95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min, 30 cycles using Taq polymerase (Roche).
  • the product was subcloned into Echo pUni/His5 R6K (Invitrogen) to generate the plasmid pR6 Kglk.
  • a PTS minus derivative (AptsHIcrr) of E. coli strain KLndh81 was obtained by P1 vir transduction using a derivative of E. coli strain NF9 as donor (Flores et al., Nature Biotechnology 14, 620-623 (1996)). The transduction replaces the operon comprising ptsH, ptsl and crr with a kanamycin antibiotic resistance marker (Levy et al., Gene 86, 27-33 (1990)) to give strain KLpts7. Plated on MacConkey (lactose ⁇ ) agar +1% glucose, KLpts7 exhibits a white colony phenotype.
  • a PCR amplification product comprising a loxP-Cat-loxP-Trc cassette and incorporating 40 bp of homology to the galP upstream region [for reference, see GenBank, Accession # U00096] to each end was generated using rtth RNA polymerase (Perkin Elmer), pR6 KgalP as the template and the primer pair SEQ ID NO:7 and SEQ ID NO:8.
  • the PCR amplified integration cassette was transformed into electro-competent KLpts7 cells containing pKD46 for integration using the lambda Red system as described in Datsenko and Wanner, supra. Selection was performed on LB plates containing 10 mg/L chloramphenicol.
  • KLpts::galP-trc plated on MacConkey (lactose ⁇ ) agar +1% glucose, exhibits a light red colony phenotype.
  • the chloramphenicol marker was removed as described by Palmeros et al., supra. The removal was confirmed by PCR analysis using the primer pair SEQ ID NO:12 and SEQ ID NO:13 (to give a 1.1 kb product) and the resulting strain was designated KLgaIP-trc.
  • a PCR amplification product comprising a loxP-Cat-loxP-Trc cassette and incorporating approximately 40 bp of homology to the glk upstream region [for reference, see GenBank, Accession # U00096] to each end was generated using rtth RNA polymerase (Perkin Elmer), pR6 Kglk as the template, and the primer pair SEQ ID NO:9 and SEQ ID NO:10.
  • the PCR amplified integration cassette was transformed into electro-competent KLgaIP-trc cells containing pKD46 for integration using the lambda Red system as described above. Selection was performed on LB plates containing 10 mg/L chloramphenicol.
  • a P1 lysate of the MG1655 ⁇ arcA::Cm strain was prepared and used to move the mutation to strain KLGG.
  • a resulting chloramphenicol resistant clone, KLGG ⁇ arcA::Cm was checked by genomic PCR to insure that the mutation was present.
  • the chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) and this strain has been designated KLGG ⁇ arcA.
  • An edd deletion [for reference, see GenBank, Accession # U00096] was obtained by replacing 1.7 kb of the coding region with a loxP-cat-loxP cassette from pLoxCat2.
  • a replacement cassette was amplified with the primer pair SEQ ID NO:16 and SEQ ID NO:17.
  • the primer SEQ ID NO:16 contains 80 bp of homology to the 5′ end of edd and 18 bp of homology to the template pLoxCat2.
  • the primer SEQ ID NO:17 contains 78 bp of homology to the 3′ end of edd and 19 bp of homology to pLoxCat2.
  • the PCR product was gel-purified and electroporated into KLGG AarcA/pKD46 competent cells.
  • Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol. The deletion of the edd gene was confirmed by PCR using primer pair SEQ ID NO:18 and SEQ ID NO:19.
  • the wild-type strain gives a 2.2 kb PCR product while the recombinant gives a characteristic 1.6 kb PCR product.
  • This strain has been designated KLGG ⁇ arcA ⁇ edd::cat.
  • the chloramphenicol marker was removed using the Cre recombinase (Palmeros et al., supra) and this strain has been designated KLGG ⁇ arcA ⁇ edd or, alternatively, FMP.
  • cells comprising ⁇ ptsHlcrr, the trc promoter replacement of the natural galP promoter, and the trc promoter replacement of the natural glk promoter (all three modifications as described in Example 4) initially exhibited slow growth. Also invariably, a subsequent selection (as described below) lead to a faster growing derivative. Glucokinase activity, assayed from cell-free extracts, was typically three-fold higher for the faster growing derivative as compared to the slower growing parent.
  • E. coli strain FMP transformed with plasmid pSYCO103, was grown in a 14 L fermenter essentially as described in Example 2. Vials for storage at ⁇ 80° C. (15% glycerol stocks) were made over the course of the fermentation. A LB plate was streaked from the aliquot taken at 33 hrs (OD 550 was 30.7 AU) and single colonies were recovered and designated “selected FMP/pSYCO103”. Single colonies were similarly obtained from strain FMP containing no plasmid and designated “selected FMP”.
  • the galP and glk genes were PCR amplified from two colonies of “selected FMP/pSYCO103” and one colony of “selected FMP” using the primer pair SEQ ID NO:12/SEQ ID NO:13 and the primer pair SEQ ID NO:14/SEQ ID NO:15, respectively. Sequence analysis on these three samples was performed using the same primers. In each case, the galP gene and promoter region remained unchanged from the parent strain while the glk gene and promoter region contained an identical, single base pair change when compared the parent strain.
  • the two isolates of “selected FMP/pSYCO103” and one isolate of “selected FMP” contained the sequence identified as SEQ ID NO:20 in the ⁇ 35 to ⁇ 10 region of trc promoter controlling glk expression compared to the corresponding parent sequence SEQ ID NO:21.
  • the strain originating from “selected FMP” from which galP and glk sequence was obtained was designated strain FMP′::Km.
  • a thawed frozen vial of FMP′::Km/pSYCO103 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture.
  • Vitamin B 12 was added to the fermenter in 16 mg boluses at 21, 40 and 40.5 h.
  • FIG. 1 shows the production of 1,3-propanediol by FMP′::Km/pSYCO103 is faster than by FMP/pSYCO103.
  • Glyceraldehyde-3-phosphate Dehydrogenase (gapA) in E. coli Strains for Production of 1,3 Propanediol from Glucose
  • GapA The level of glyceraldehyde-3-phosphate dehydrogenase, GapA, was decreased by replacing the ATG start codon of the gapA gene with a GTG or TTG start codon.
  • E. coli gapA gene plus upstream and downstream flanking sequence was amplified by PCR from E. coli strain MG1655 using primer pair SEQ ID NO:22 and SEQ ID NO:23.
  • the PCR product was cloned directly from the PCR reaction into pCR-BluntII-TOPO (Invitrogen) to form pDT50.
  • the plasmids pDT50 and pLitmus 28 (New England Biolabs, Inc.) were digested with SphI and BamH I and the gapA gene fragment and vector, respectively, were gel-purified and ligated.
  • the resulting plasmid, pDT51 was transformed into E. coli TOP10 (Invitrogen).
  • the gapA mutant plasmids were constructed using the Stratagene QuickChange 1-Day Site-Directed Mutagenesis Method (Stratagene, La Jolla, Calif.).
  • the template plasmid, pDT51 was mixed with either the primer pair SEQ ID NO:24 and SEQ ID NO:25 to create the GTG mutation or the primer pair SEQ ID NO:26 and SEQ ID NO:27 to create the TTG mutation.
  • the reactions were digested with Dpnl to remove the template plasmid and leave only the amplified plasmids.
  • the plasmids were then transformed into E. coli TOP10 (Invitrogen).
  • the gapA-GTG and gapA-TTG constructs were PCR amplified using primer pair SEQ ID NO:22 and SEQ ID NO:23.
  • the PCR products were electroporated into the gapA knockout strain, KLP23A112.
  • E. coli strain KLP23A112 was constructed by transducing KLP23 with a P1 phage lysate obtained from E. coli DS112 ( E. coli Genetic Stock Center), a gapA deletion strain containing a CmR marker. Recombinants were selected for growth on LB plates without added glucose and sensitivity to chloramphenicol. Sequencing confirmed the successful integration of the GTG and TTG mutations.
  • the mutated strains were named KLPAGTG and KLPATTG, respectively. Measurement of the GapA activities in the KLPAGTG and KLPATTG strains showed that the GapA levels were 4% and ⁇ 1% of the control strain KLP23, respectively.
  • Primer SEQ ID NO:30 contains 59 bp of homology to upstream gapA sequence and 21 bp of homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells to give MG1655 1.5gapA::Cm.
  • Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol. Successful integration of the cassette replaces the region 34-258 bp upstream of the gapA ATG start codon with a FRT-CmR-FRT-short 1.5 GI promoter cassette.
  • a P1 phage lysate was prepared and used to move the mutation to FMP′::Km. This strain was designated FMP′::Km 1.5gapA::Cm.
  • the short 1.5 GI gapA promoter in MG1655 1.5gapA::Cm was replaced with the short 1.20 GI promoter (SEQ ID NO:31) or the short 1.6 GI promoter (SEQ ID NO:32).
  • a replacement cassette was PCR amplified with primer pair SEQ ID NO:33 and SEQ ID NO:34 using genomic DNA from MG1655 1.5gapA::Cm as template.
  • Primer SEQ ID NO:33 contains 24 bp of homology to the gapA upstream region.
  • Primer SEQ ID NO:34 contains homology to the gapA upstream region in MG1655 1.5gapA::Cm and contains the short 1.20 GI promoter.
  • a replacement cassette was PCR amplified with primer pair SEQ ID NO:33 and SEQ ID NO:35 using genomic DNA from MG1655 1.5gapA::Cm as template.
  • the primer SEQ ID NO:35 contains homology to the gapA upstream region in MG1655 1.5gapA::Cm and contains the short 1.6 GI promoter.
  • the short 1.20 GI promoter replacement cassette and the short 1.6 GI promoter replacement cassette were used to replace the natural gapA promoter of MG1655 as described above to give strains MG1655 1.20gapA::Cm and MG1655 1.6gapA::Cm, respectively.
  • MG1655 1.20gapA::Cm and MG1 655 1.6gapA::Cm were used to replace the natural gapA promoter of strain FMP′::Km (using P1 tranduction as described above) to give strains FMP′::Km 1.20gapA::Cm and FMP′::Km 1.6gapA::Cm, respectively.
  • Glyceraldehyde-3-phosphate dehydrogenase activities were determined using cell-free extracts prepared from the strains FMP′::Km 1.20gapA::Cm, FMP′::Km 1.5gapA::Cm, FMP′::Km 1.6gapA::Cm and FMP′::Km as control.
  • the values obtained, compared to that of control, were 10%, 25% and 140% for strains FMP′::Km 1.20gapA::Cm, FMP′::Km 1.5gapA::Cm, FMP′::Km 1.6gapA::Cm, respectively.
  • the chloramphenicol marker was removed from strain FMP′::Km 1.5gapA::Cm (as described in the General Methods section) to give strains FMP′::Km 1.5gapA.
  • kanamycin marker introduced into FMP′::Km 1.5gapA as a consequence of making KLndh81 PTS minus ( ⁇ ptsHlcrr) was replaced with a removable FRT-Cm-FRT cassette by P1 transduction from MG1655 ⁇ ptsHlcrr::Cm.
  • a ptsHlcrr deletion in MG1655 was made with a replacement cassette amplified with primer pair SEQ ID NO:54 and SEQ ID NO:55 using pKD3 as template.
  • the primer SEQ ID NO:54 contains 78 bp of homology to the remaining region of ptsH left in the chromosome of strain FMP′::Km 1.5gapA and 20 bp of homology to pKD3.
  • the primer SEQ ID NO:55 contains 77 bp of homology to the remaining region of crr in strain FMP′::Km 1.5gapA and 20 bp of homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with chloramphenicol 12.5 mg/L. PCR analysis confirmed the integration of the cassette.
  • Strain FMP′ 1.5gapA was transformed with the plasmid pSYCO106. Production of 1,3-propanediol and glycerol was determined in 14 L fermenters as described in General Methods with the following differences in the control parameters for the fermenter. A thawed frozen vial of FMP′ 1.5gap/pSYCO106 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture. Vitamin B 12 was added to the fermenter in 16 mg boluses prior to inoculation and at 28 hrs. Final 1,3-propanediol concentration was 129 g/L and the mass yield was 40.2%.
  • the mgsA deletions [for reference, see GenBank, Accession # U00096] were made by replacing 0.4 kb of the coding region with the FRT-Kan-FRT cassette of pKD4.
  • a replacement cassette was PCR amplified with the primer pair SEQ ID NO:36 and SEQ ID NO:37 using pKD4 as the template.
  • the primer SEQ ID NO:36 contains 40 bp of homology to the 5′ end of mgsA and 20 bp of homology to the template DNA, pKD4.
  • the primer SEQ ID NO:37 contains 40 bp of homology to the 3′ end of mgsA and 20 bp of homology to pKD4.
  • the PCR product was gel-purified and electroporated into MG1655/pKD46 competent cells.
  • Recombinant strains were selected on LB plates with 12.5 mg/L of kanamycin. The deletion of the mgsA gene was confirmed by PCR, using the primer pair SEQ ID NO:38 and SEQ ID NO:39.
  • the wild-type strain gives a 1.3 kb PCR product while the recombinant strain gives a characteristic 2.4 kb PCR product.
  • This strain has been designated MG1655 ⁇ mgsA::kan.
  • a P1 phage lysate was prepared and used to move the mutation into FMP′ 1.5gapA (Example 8).
  • the kanamycin resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) and this strain has been designated FMP′ 1.5gapA AmgsA.
  • FMP′ 1.5gapA ⁇ mgsA and its parent were transformed with the plasmid pSYCO106 to give FMP′1.5gapA ⁇ mgsA/pSYCO106 and FMP′ 1.5gapA/pSYCO106, respectively.
  • Strain FMP′ 1.5gapA ⁇ mgsA was transformed with the plasmid pSYCO106. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermenters. A single colony from a fresh plate (LA with 50 mg/L spectinomycin) of FMP′ 1.5gapA ⁇ mgsA pSYCO106 was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to prepare the pre-culture. After incubation at 34° C.
  • PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol to give MG1655 1.6 ppc::Cm. Successful integration of the cassette replaces the region 90 to 148 bp upstream of the ppc ATG start [for reference, see GenBank, Accession # U00096] with a FRT-CmR-FRT-short 1.6 GI promoter cassette. Integration into the upstream ppc region was confirmed by primer pair SEQ ID NO:40 and SEQ ID NO:41.
  • the wild-type strain gives a 0.2 kb PCR product while the recombinant strain gives a characteristic 1.2 kb PCR product.
  • This PCR product was sequenced using primer SEQ ID NO:42, which indicated that the promoter replacement effectively occurred.
  • a P1 phage lysate was prepared and used to move the mutation to strain FMP′1.5gap ⁇ mgsA. This strain was designated FMP′1.5gap ⁇ mgs 1.6 ppc::Cm.
  • the chloramphenicol-resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra), and the resulting strain was electrotransformed with plasmid pSYCO106 to give FMP′1.5gap ⁇ mgs 1.6 ppc/pSYCO106.
  • Phosphoenolpyruvate carboxylase (Ppc) activity was measured from cell-free extracts obtained from the shake flasks described immediately above. Aliquots of cells were harvested in mid-log phase, broken by two passages through a French press cell, centrifuged for 15 15 min at 14,000 rpm, and ultracentrifuged 1 hr at 50,000 rpm. The supernatant was removed and used as a source of proteins. Specific activities of PPC are reported in Table 8 below. The replacement of the natural ppc promoter with the short 1.6 GI promoter increased the Ppc enzyme activity three-fold.
  • the genes yciK and btuR are present within a single operon in E. coli .
  • Replacement of the natural yciK-btuR promoter with the synthetic short 1.6 GI promoter was made by inserting a 1.3 kb cassette, upstream of yciK-btuR.
  • the replacement cassette, containing FRT-CmR-FRT and an engineered promoter, was amplified by PCR with the primer pair SEQ ID NO:43 and SEQ ID NO:44 using pKD13 as the template.
  • Primer SEQ ID NO:43 contains 70 bp of homology to upstream yciK-btuR sequence and 20 bp of homology to template pKD13.
  • Primer SEQ ID NO:44 contains 30 bp of homology to upstream yciK-btuR sequence, contains the short 1.6 GI promoter sequence and contains 20 bp of homology to pKD13.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 25.0 mg/L kanamycin to give MG1655 1.6yciK-btuR::Km.
  • Primer SEQ ID NO:48 incorporates 41 bp of homology (with a 1 bp deletion) to upstream yqhD sequence, 40 bp of homology to the short 1.6 GI promoter sequence, and 19 bp of homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol to give MG1655 1.6yqhD::Cm.
  • Vitamin B 12 was added to the fermenter in a single 16 mg bolus at 20.6 hrs elapsed fermentation time.
  • Final 1,3-propanediol concentration was 113.3 g/L and the mass yield was 48.8%.
  • the pta-ackA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 3.3 kb of the coding region with the FRT-CmR-FRT cassette of pKD3.
  • the replacement cassette was amplified with the primer pair SEQ ID NO:50 and SEQ ID NO:51 using pKD3 as the template.
  • the primer SEQ ID NO:50 contains 80 bp of homology to the 5′ end of pta and 20 bp of homology to the template DNA, pKD3.
  • the primer SEQ ID NO:51 contains 80 bp of homology to the 3′ end of ackA and 20 bp of homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells.
  • Recombinant strains were selected on LB plates with 12.5 mg/L of chloramphenicol to give strain MG1655 ⁇ ackA-pta::Cm.
  • the deletion of the pta-ackA genes was confirmed by PCR, using the primer pair SEQ ID NO:52 and SEQ ID NO:53.
  • the wild-type strain gives a 3.8 kb PCR product while the recombinant strain gives a characteristic 1.6 kb PCR product.
  • a P1 phage lysate was prepared and used to pass the mutation to strain Triple 1.6btuR 1.6yqhD to form strain Triple 1.6btuR 1.6yqhD AackA-pta::Cm.
  • the chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to give Triple 1.6btuR 1.6yqhD AackA-pta (renamed TripleTriple (TT)).
  • the Triple 1.6btuR 1.6yqhD and TT strains were electrotransformed with plasmid pSYCO109.
  • a pre-culture of TT/pSYCO109 was grown in 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to an OD550 of approximately 1 AU.
  • a seed fermenter prepared as described was inoculated with 10.8 mL of that culture. After 30.5 hrs of fermentation time, 1.2 L of the culture was transferred to a production fermenter. This fermenter received a single 16 mg bolus of vitamin B 12 1 hr after inoculation. The final concentration of 1,3-propanediol in a typical fermentation was 114 g/L and the yield was 48%.
  • Duplicate shake flasks cultures were grown with strains Triple btuR 1.6 yqhD, pSYCO109, and TT pSYCO109. After incubating one colony for 10 hr in LB+50 mg/L spectinomycin, 100 mL of culture were transferred in 30 mL TM2 medium with 2% glucose and with or without 50 ppm spectinomycin (day 1). In order to study the stability of the yield, a 100 mL volume of the day 1 cultures was transferred after 24 hr to a fresh volume of 30 mL TM2 media containing 2% glucose with or without 50 ppm spectinomycin. This was repeated 4 times.
  • An aldA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 1.3 kb of the coding region with the FRT-CmR-FRT cassette of pKD3.
  • the cassette was amplified with the primer pair SEQ ID NO:57 and SEQ ID NO:58 using pKD3 as the template.
  • the primer SEQ ID NO:57 contains 80 bp of homology to the 5′ end of aldA and 20 bp of homology to the template DNA, pKD3.
  • the primer SEQ ID NO:58 contains 80 bp of homology to the 3′ end of aldA and 20 bp of homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells.
  • Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol.
  • the deletion of the aldA gene was confirmed by PCR, using the primer pair SEQ ID NO:59 and SEQ ID NO:60.
  • the wild-type strain gives a 2.0 kb PCR product while the recombinant strain gives a characteristic 1.8 kb PCR product.
  • a P1 lysate of that strain was prepared and used to move the mutation to the strain TT to form the TT ⁇ aldA::Cm strain.
  • the chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to create TT aldA.
  • An aldB deletion [for reference, see GenBank, Accession # U00096] was made by replacing 1.5 kb of the coding region with the FRT-CmR-FRT cassette of the pKD3.
  • a replacement cassette was amplified with the primer pair SEQ ID NO:61 and SEQ ID NO:62 using pKD3 as the template.
  • the primer SEQ ID NO:61 contains 80 bp of homology to the 5′-end of aldB and 20 bp of homology to pKD3.
  • Primer SEQ ID NO:62 contains 80 bp of homology to the 3′ end of aldB and 20 bp homology to pKD3.
  • the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells.
  • Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol.
  • the deletion of the aldB gene was confirmed by PCR, using the primer pair SEQ ID NO:63 and SEQ ID NO:64.
  • the wild-type strain gives a 1.5 kb PCR product while the recombinant strain gives a characteristic 1.1 kb PCR product.
  • a P1 lysate was prepared and used to move the mutation to the TT strain to form the TT ⁇ aldB::Cm strain.
  • a chloramphenicol-resistant clone was checked by genomic PCR with the primer pair SEQ ID NO:63 and SEQ ID NO:64 to insure that the mutation was present.
  • Strain FMP′ 1.5gapA was transformed with the plasmid pSYCO106. Production of glycerol was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermentation. A thawed frozen vial of FMP′ 1.5gap/pSYCOl06 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture. No vitamin B 12 was added to the fermenter. A typical fermentation resulted in the production of 202 g/L glycerol with a molar yield of 115%.

Abstract

The present invention provides a microorganism useful for biologically producing 1,3-propanediol from a fermentable carbon source at higher yield than was previously known. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence to produce 1,3-propanediol. The invention provides a microorganism with disruptions in specified genes and alterations in the expression levels of specified genes that is useful in a higher yielding process to produce 1,3-propanediol.

Description

  • This application claims the benefit of U.S. Provisional Application No. 60/416,192, filed Oct. 4, 2003. [0001]
    • FIELD OF INVENTION
  • This invention comprises a process for the bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. [0002]
    • BACKGROUND
  • 1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. [0003]
  • A variety of chemical routes to 1,3-propanediol are known. For example ethylene oxide may be converted to 1,3-propanediol over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid, by the catalytic solution phase hydration of acrolein followed by reduction, or from compounds such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from group VIII of the periodic table. Although it is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants. [0004]
  • It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two step, enzyme catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water, Equation 1. In the second step, 3-HPA is reduced to 1,3-propanediol by a NAD[0005] +-linked oxidoreductase, Equation 2. The 1,3-propanediol is not metabolized further and, as a result,
  • Glycerol→3-HPA+H2O  (Equation 1)
  • 3-HPA+NADH+H+→1,3-Propanediol+NAD+  (Equation 2)
  • accumulates in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced β-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD+). [0006]
  • In [0007] Klebsiella pneumonia, Citrobacter freundii, and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase (dhaB1-3 or dhaB, C and E) are located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase (dhaT). Although the genetic organization differs somewhat among these microorganisms, these genes are clustered in a group which also comprises orfx and orfZ (genes encoding a dehydratase reactivation factor for glycerol dehydratase), as well as orfY and orfW (genes of unknown function). The specific 1,3-propanediol oxidoreductases (dhaTs) of these microorganisms are known to belong to the family of type III alcohol dehydrogenases; each exhibits a conserved iron-binding motif and has a preference for the NAD+/NADH linked interconversion of 1,3-propanediol and 3-HPA. However, the NAD+/NADH linked interconversion of 1,3-propanediol and 3-HPA is also catalyzed by alcohol dehydrogenases which are not specifically linked to dehydratase enzymes (for example, horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)), albeit with less efficient kinetic parameters. Glycerol dehydratase (E.C. 4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinct enzymes that are encoded by distinct genes. Diol dehydratase genes from Klebsiella oxytoca and Salmonella typhimurium are similar to glycerol dehydratase genes and are clustered in a group which comprises genes analogous to orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999); Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); GenBank AF026270).
  • The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions, in e.g., strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD[0008] +-(or NADP+-) linked glycerol dehydrogenase, Equation 3. The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4),
  • Glycerol+NAD+→DHA+NADH+H+  (Equation 3)
  • DHA+ATP→DHAP+ADP  (Equation 4)
  • becomes available for biosynthesis and for supporting ATP generation via e.g., glycolysis. In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH. [0009]
  • In [0010] Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulon, in Klebsiella pneumoniae and Citrobacter freundii, also encompasses a gene encoding a transcriptional activator protein (dhaR). The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.
  • Neither the chemical nor biological methods described above for the production of 1,3-propanediol are well suited for industrial scale production since the chemical processes are energy intensive and the biological processes are limited to relatively low titer from the expensive starting material, glycerol. These drawbacks could be overcome with a method requiring low energy input and an inexpensive starting material such as carbohydrates or sugars, or by increasing the metabolic efficiency of a glycerol process. Development of either method will require the ability to manipulate the genetic machinery responsible for the conversion of sugars to glycerol and glycerol to 1,3-propanediol. [0011]
  • Biological processes for the preparation of glycerol are known. The overwhelming majority of glycerol producers are yeasts but some bacteria, other fungi, and algae are also known. Both bacteria and yeasts produce glycerol by converting glucose or other carbohydrates through the fructose-1,6-bisphosphate pathway in glycolysis or the Embden Meyerhof Parnas pathway. Dihydroxyacetone phosphate is converted to glycerol-3-phosphate by the action of glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphate is converted to glycerol by the action of glycerol-3-phosphatase. [0012]
  • The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from [0013] S. diastaticus (Wang et al., J. Bact. 176, 7091-7095 (1994)). The DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al. (supra) recognize that DAR1 is regulated by the cellular osmotic environment but do not suggest how the gene might be used to enhance 1,3-propanediol production in a recombinant microorganism.
  • Other glycerol-3-phosphate dehydrogenase enzymes have been isolated: for example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from [0014] Saccharomyces cerevisiae (Larason et al., Mol. Microbiol. 10, 1101 (1993)) and Albertyn et al. (Mol. Cell. Biol. 14, 4135 (1994)) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Like Wang et al. (supra), both Albertyn et al. and Larason et al. recognize the osmosensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of 1,3-propanediol in a recombinant microorganism.
  • As with G3PDH, glycerol-3-phosphatase has been isolated from [0015] Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)). Like the genes encoding G3PDH, it appears that GPP2 is osmosensitive.
  • WO 9634961 and Hernandez-Montalvo et al. ([0016] Appl. Microbiol. Biotechnol. 57:186-191 (2001) describe E. coli strains that have “PTS” minus/glucose plus phenotypes. EP 1170376 A1 discloses deletion of a gene for NADH dehydratase II to improve energy efficiency. WO 2001016346 describes the utility of “aldehyde dehydrogenase A” and “aldehyde dehydrogenase B” for the production of 3-hydroxypropionic acid.
  • WO 9635796 (U.S. Pat. No. 5,686,276, E. I. du Pont de Nemours and Company (“DuPont”)) discloses a method for the production of 1,3-propanediol from a carbon substrate other than glycerol or dihydroxyacetone (especially, e.g., glucose), using a single microorganism comprising a dehydratase activity. WO 9928480 (DuPont) discloses a similar method with advantages derived from expressing exogenous activities of one or both of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase while disrupting one or both of endogenous activities glycerol kinase and glycerol dehydrogenase. WO 9821339 (U.S. Pat. No. 6,013,494, DuPont) describes a process for the production of 1,3-propanediol using a single microorganism comprising exogenous glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, dehydratase, and 1,3-propanediol oxidoreductase (e.g., dhaT). WO 9821341 (U.S. Pat. No. 6,136,576, DuPont) discloses a method for the production of 1,3-propanediol comprising a recombinant microorganism further comprising a dehydratase and protein X (later identified as being a dehydratase reactivation factor peptide). WO 2001012833 (DuPont) describes an improvement to the process where a significant increase in titer (grams product per liter) is obtained by virtue of a non-specific catalytic activity (distinguished from 1,3-propanediol oxidoreductase encoded by dhaT) to convert 3-hydroxypropionaldehyde to 1,3-propanediol. U.S. Ser. No. 10/420,587 (2003) (U.S. 60/374,931 (2002)DuPont)) discloses vectors and plasmids useful for the production of 1,3-propanediol. The DuPont applications are incorporated by reference in the instant specification as though set forth in their entirety herein. [0017]
  • The biological production of 1,3-propanediol requires glycerol as an intermediate substrate for a two-step sequential reaction in which a dehydratase enzyme (typically a coenzyme B[0018] 12-dependent dehydratase) converts glycerol to 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH- (or NADPH-) dependent oxidoreductase. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence for the production of 1,3-propanediol.
  • A specific deficiency in the biological processes leading to the production of 1,3-propanediol from glucose has been the low yield of the product achieved via fermentation. WO 2001012833 (DuPont) describes weight yields of 1,3-propanediol from glucose within the range of 24% and 35%. The problem that remains to be solved is how to biologically produce 1,3-propanediol, with high yield and by a single microorganism, from an inexpensive carbon substrate such as glucose or other sugars. [0019]
    • SUMMARY OF THE INVENTION
  • Applicants have solved the stated problem. The present invention provides for bioconverting a fermentable carbon source to 1,3-propanediol at higher yield than previously obtained and with the use of a single microorganism. The yield obtained is greater than 35%, and preferably greater than 40%. Glucose is used as a model substrate and [0020] Escherichia coli is used as the model host microorganism with the useful genetic modifications and disruptions detailed herein.
  • Applicants have provided an [0021] E. coli strain comprising:
  • a) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system preventing expression of active PEP-glucose phosphotransferase system proteins; [0022]
  • b) an up regulated endogenous galP gene encoding active galactose-proton symporter; [0023]
  • c) an up regulated endogenous glk gene encoding active glucokinase; and [0024]
  • d) a down regulated endogenous gapA gene encoding active glycerolaldehyde 3-phosphate dehydrogenase. [0025]
  • Applicants have also provided an [0026] E. coli strain described above wherein the disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprises one or more of
  • a1) a disrupted endogenous ptsH gene preventing expression of active phosphocarrier protein; [0027]
  • a2) a disrupted endogenous ptsl gene preventing expression of active phosphoenolpyruvate-protein phosphotransferase; and [0028]
  • a3) a disrupted endogenous crr gene preventing expression of active glucose-specific IIA component. [0029]
  • The [0030] E. coli embodiments described above can further comprise one or more of
  • e) a disrupted endogenous arcA gene preventing expression of active aerobic respiration control protein; [0031]
  • f) an up regulated endogenous ppc gene encoding active phosphoenolpyruvate carboxylase; [0032]
  • g) an up regulated endogenous btuR gene encoding active cob(I)alamin adenosyltransferase; and [0033]
  • h) an up regulated yqhD gene encoding active alcohol dehydrogenase. [0034]
  • The [0035] E. coli embodiments described above can further comprise one or more of
  • i) a disrupted endogenous mgsA gene preventing the expression of active methylglyoxal synthase; [0036]
  • j) a disrupted endogenous ackA gene preventing the expression of active acetate kinase; [0037]
  • k) a disrupted endogenous pta gene preventing the expression of active phosphotrasacetylase; [0038]
  • l) a disrupted endogenous aldA gene preventing the expression of active aldehyde dehydrogenase A; and [0039]
  • m) a disrupted endogenous aldB gene preventing the expression of active aldehyde dehydrogenase B. [0040]
  • The [0041] E. coli embodiments described above can further comprise one or more of:
  • n) a disrupted endogenous edd gene preventing expression of active phosphogluconate dehydratase; [0042]
  • o) a disrupted endogenous glpk gene preventing expression of active glycerol kinase; and [0043]
  • p) a disrupted endogenous gldA gene preventing expression of active NADH-dependent glycerol dehydrogenase. [0044]
  • Additionally, 1,3-propanediol can be bioproduced by contacting an [0045] E. coli strain described herein with a suitable carbon substrate such as glucose under suitable conditions for fermentation. In addition, 1,3-propanediol can be bioproduced by contacting an E. coli strain described herein, the E. coli strain further comprising an active:
  • (i) glycerol-3-phosphate dehydrogenase; [0046]
  • (ii) glycerol-3-phosphatase; [0047]
  • (iii) dehydratase; and [0048]
  • (iv) dehydratase reactivating activity; [0049]
  • with a suitable carbon substrate under suitable conditions. [0050]
  • Additionally, any of the embodiments disclosed above may also include the constructs pSYCO101, pSYCO103, pSYCO106, pSYCO109 or their corresponding nucleotide sequences SEQ ID NOs:65, 66, 67, or 68. [0051]
    • BRIEF DESCRIPTION OF THE DRAWINGS SEQUENCE DESCRIPTIONS, AND BIOLOGICAL DEPOSITS
  • The invention can be more fully understood from the following detailed description, the FIG. 1, the accompanying sequence listing and descriptions, and biological deposits that form parts of this application. [0052]
  • FIG. 1 shows 1,3-propanediol production compared as between two fermentations run essentially as described in GENERAL METHODS. In one case, the strain used was FMP′::Km/pSYCO103. In the other case, the strain used was FMP/pSYCO103. [0053]
  • The 68 sequence descriptions and the sequence listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and will be consistent with World Intellectual Property Organization (WIPO) Standard ST2.5 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administration Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in [0054] Nucleic Acids Res. 13, 3021-3030 (1985) and in the Biochemical Journal 219, 345-373 (1984) which are herein incorporated by reference.
  • SEQ ID NO:1 is the partial nucleotide sequence of pLoxCat27 encoding the loxP511-Cat-loxP511 cassette. [0055]
  • SEQ ID NO:2-3 are oligonucleotide primers used to construct the arcA disruption. [0056]
  • SEQ ID NOs:4-5 are oligonucleotide primers used to confirm disruption of arcA. [0057]
  • SEQ ID NO:6 is the partial nucleotide sequence of pLoxCat1 encoding the loxP-Cat-loxP cassette. [0058]
  • SEQ ID NOs:7-8 are oligonucleotide primers used to construct pR6 KgalP, the template plasmid for trc promoter replacement of the chromosomal galP promoter. [0059]
  • SEQ ID NOs:9-10 are oligonucleotide primers used to construct pR6 Kglk, the template plasmid for trc promoter replacement of the chromosomal glk promoter. [0060]
  • SEQ ID NO:11 is the nucleotide sequence of the loxP-Cat-loxP-Trc cassette. [0061]
  • SEQ ID NOs:12-13 are oligonucleotide primers used to confirm integration of SEQ ID NO:11 for replacement of the chromosomal galP promoter. [0062]
  • SEQ ID NOs:14-15 are oligonucleotide primers used to confirm integration of SEQ ID NO:11 for replacement of the chromosomal glk promoter. [0063]
  • SEQ ID NOs:16-17 are oligonucleotide primers used to construct the edd disruption. [0064]
  • SEQ ID NOs:18-19 are oligonucleotide primers used to confirm disruption of edd. [0065]
  • SEQ ID NOs:20 is the nucleotide sequence for the selected trc promoter controlling glk expression. [0066]
  • SEQ ID NOs:21 is the partial nucleotide sequence for the standard trc promoter. [0067]
  • SEQ ID NOs:22-23 are the oligonucleotide primers used for amplification of gapA. [0068]
  • SEQ ID NOs:24-25 are the oligonucleotide primers used to alter the start codon of gapA to GTG. [0069]
  • SEQ ID NOs:26-27 are the oligonucleotide primers used to alter the start codon of gapA to TTG. [0070]
  • SEQ ID NO:28 is the nucleotide sequence for the short 1.5 GI promoter. [0071]
  • SEQ ID NOs:29-30 are oligonucleotide primers used for replacement of the chromosomal gapA promoter with the short 1.5 GI promoter. [0072]
  • SEQ ID NO:31 is the nucleotide sequence for the short 1.20 GI promoter. [0073]
  • SEQ ID NO:32 is the nucleotide sequence for the short 1.6 GI promoter. [0074]
  • SEQ ID NOs:33-34 are oligonucleotide primers used for replacement of the chromosomal gapA promoter with the short 1.20 GI promoter. [0075]
  • SEQ ID NO:35 is the oligonucleotide primer with SEQ ID NO 33 that is used for replacement of the chromosomal gapA promoter with the short 1.6 GI promoter. [0076]
  • SEQ ID NOs:36-37 are oligonucleotide primers used to construct the mgsA disruption. [0077]
  • SEQ ID NOs:38-39 are oligonucleotide primers used to confirm disruption of mgsA. [0078]
  • SEQ ID NOs:40-41 are oligonucleotide primers used for replacement of the chromosomal ppc promoter with the short 1.6 GI promoter. [0079]
  • SEQ ID NO:42 is an oligonucleotide primer used to confirm replacement of the ppc promoter. [0080]
  • SEQ ID NOs:4344 are oligonucleotide primers used for replacement of the chromosomal yciK-btuR promoter with the short 1.6 GI promoter. [0081]
  • SEQ ID NOs:45-46 are oligonucleotide primers used to confirm replacement of the yciK-btuR promoter. [0082]
  • SEQ ID NOs:47-48 are oligonucleotide primers used for replacement of the chromosomal yqhD promoter with the short 1.6 GI promoter. [0083]
  • SEQ ID NO:49 is an oligonucleotide primer used to confirm replacement of the yqhD promoter. [0084]
  • SEQ ID NOs:50-51 are oligonucleotide primers used to construct the pta-ackA disruption. [0085]
  • SEQ ID NOs:52-53 are oligonucleotide primers used to confirm disruption of pta-ackA. [0086]
  • SEQ ID NOs:54-55 are oligonucleotide primers used to construct the ptsHIcrr disruption. [0087]
  • SEQ ID NO:56 is an oligonucleotide primer used to confirm disruption of ptsHlcrr. [0088]
  • SEQ ID NOs:57-58 are oligonucleotide primers used to construct the aldA disruption. [0089]
  • SEQ ID NOs:59-60 are oligonucleotide primers used to confirm disruption of aldA. [0090]
  • SEQ ID NOs:61-62 are oligonucleotide primers used to construct the aldB disruption. [0091]
  • SEQ ID NOs:63-64 are oligonucleotide primers used to confirm disruption of aldB. [0092]
  • SEQ ID NO:65 is the nucleotide sequence for the pSYCO101 plasmid. [0093]
  • SEQ ID NO:66 is the nucleotide sequence for the pSYCO103 plasmid. [0094]
  • SEQ ID NO:67 is the nucleotide sequence for the pSYCO106 plasmid. [0095]
  • SEQ ID NO:68 is the nucleotide sequence for the pSYCO109 plasmid. [0096]
  • Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. [0097]
    Depositor Identification Int'l Depository
    Reference Designation Date of Deposit
    Transformed E. coli DH5α ATCC 69789 18 Apr. 1995
    containing a portion of the Klebsiella
    genome encoding the glycerol
    dehydratase enzyme
    Transformed E. coli DH5α ATCC 69790 18 Apr. 1995
    containing cosmid pKP4 containing
    a portion of Klebsiella genome
    encoding a diol dehydratase enzyme
    E. coli MSP33.6 ATCC 98598 25 Nov. 1997
    glpK mutant E. coli RJF10m ATCC 98597 25 Nov. 1997
    Escherichia coli: RJ8n ATCC PTA4216 9 Apr. 2002
    Escherichia coli: FMP'::Km ATCC PTA-4732 28 Sep. 2002
  • The deposit(s) will be maintained in the indicated international depository for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action. [0098]
  • As used herein, “ATCC” refers to the American Type Culture Collection international depository located 10801 University Blvd., Manassas, Va. 20110-2209 U.S.A. The “ATCC No.” is the accession number to cultures on deposit with the ATCC.[0099]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides for an improved process for bioconverting a fermentable carbon source directly to 1,3-propanediol using a single microorganism. The method is characterized by improved 1,3-propanediol yield at levels not previously obtained. [0100]
  • The disclosed production host strains have been engineered to maximize the metabolic efficiency of the pathway by incorporating various deletion mutations that prevent the diversion of carbon to non-productive compounds. It is contemplated that transformations and mutations can be combined so as to control particular enzyme activities for the enhancement of 1,3-propanediol production. Thus, it is within the scope of the present invention to anticipate modifications of a whole cell catalyst which lead to an increased production of 1,3-propanediol. [0101]
  • Terms And Definitions [0102]
  • The invention can be more fully understood with reference to the following terms and definitions used in the claims and specifications. [0103]
  • Genes that are Deleted: [0104]
  • The terms “NADH dehydrogenase II”, “NDH II” and “Ndh” refer to the type II NADH dehydrogenase, a protein that catalyzed the conversion of ubiquinone-8+NADH+H[0105] + to ubiquinol-8+NAD+. Typical of NADH dehydrogenase II is EC 1.6.99.3. NADH dehydrogenase II is encoded by ndh in E. coli.
  • The terms “aerobic respiration control protein” and “ArcA” refer to a global regulatory protein. The aerobic respiration control protein is encoded by arcA in [0106] E. coli.
  • The terms “phosphogluconate dehydratase” and “Edd” refer to a protein that catalyzed the conversion of 6-phospho-gluconate to 2-keto-3-deoxy-6-phospho-gluconate+H[0107] 2O. Typical of phosphogluconate dehydratase is EC 4.2.1.12. Phosphogluconate dehydratase is encoded by edd in E. coli.
  • The terms “phosphocarrier protein HPr” and “PtsH” refer to the phosphocarrier protein encoded by ptsH in [0108] E. coli. The terms “phosphoenolpyruvate-protein phosphotransferase” and “Ptsl” refer to the phosphotransferase, EC 2.7.3.9, encoded by ptsl in E. coli. The terms “PTS system”, “glucose-specific IIA component”, and “Crr” refer to EC 2.7.1.69, encoded by crr in E. coli. PtsH, Ptsl, and Crr comprise the PTS system.
  • The term “phosphoenolpyruvate-sugar phosphotransferase system”, “PTS system”, or “PTS” refers to the phosphoenolpyruvate-dependent sugar uptake system. [0109]
  • The terms “methylglyoxal synthase” and “MgsA” refer to a protein that catalyzed the conversion of dihydroxy-acetone-phosphate to methyl-glyoxal+phosphate. Typical of methylglyoxal synthase is EC 4.2.3.3. Methylglyoxal synthase is encoded by mgsA in [0110] E. coli.
  • The terms “aldehyde dehydrogenase A” and “AldA” refer to a protein that catalyzed the conversion of H[0111] 2O+NAD++aldehyde to NADH+alcohol. Typical of aldehyde dehydrogenase A is EC 1.2.1.22. Aldehyde dehydrogenase A is encoded by aldA in E. coli.
  • The terms “aldehyde dehydrogenase B” and “AldB” refer to a protein that catalyzed the conversion of H[0112] 2O+NAD++aldehyde to NADH+alcohol. Typical of aldehyde dehydrogenase B is EC 1.2.1.22. Aldehyde dehydrogenase B is encoded by aldB in E. coli.
  • Genes whose Expression has been Modified: [0113]
  • The terms “galactose-proton symporter” and “GalP” refer to a protein that catalyses the transport of a sugar and a proton from the periplasm to the cytoplasm. D-glucose is a preferred substrate for GalP. Galactose-proton symporter is encoded by galP in [0114] E. coli.
  • The terms “glucokinase” and “Glk” refer to a protein that catalyses the conversion of D-glucose+ATP to glucose-6-phosphate+ADP. Typical of glucokinase is EC 2.7.1.2. Glucokinase is encoded by glk in [0115] E. coli.
  • The terms “glyceraldehyde 3-phosphate dehydrogenase” and “GapA” refer to a protein that catalyses the conversion of glyceraldehyde-3-phosphate+phosphate+NAD[0116] + to 3-phospho-D-glyceroyl-phosphate+NADH+H+. Typical of glyceraldehyde 3-phosphate dehydrogenase is EC 1.2.1.12. Glyceraldehyde 3-phosphate dehydrogenase is encoded by gapA in E. coli.
  • The terms “phosphoenolpyruvate carboxylase” and “Ppc” refer to a protein that catalyses the conversion of phosphoenolpyruvate+H[0117] 2O+CO2 to phosphate+oxaloacetic acid. Typical of phosphoenolpyruvate carboxylase is EC 4.1.1.31. Phosphoenolpyruvate carboxylase is encoded by ppc in E. coli.
  • The term “YciK” refers to a putative enzyme encoded by yciK which is translationally coupled to btuR, the gene encoding Cob(I)alamin adenosyltransferase in [0118] Escherichia coli.
  • The term “cob(I)alamin adenosyltransferase” refers to an enzyme responsible for the transfer of a deoxyadenosyl moiety from ATP to the reduced corrinoid. Typical of cob(I)alamin adenosyltransferase is EC 2.5.1.17. Cob(I)alamin adenosyltransferase is encoded by the gene “btuR” (GenBank M21528) in [0119] Escherichia coli, “cobA” (GenBank L08890) in Salmonella typhimurium, and “cobO” (GenBank M62866) in Pseudomonas denitrificans.
  • Additional Definitions: [0120]
  • The term “short 1.20 GI promoter” refers to SEQ ID NO:31. The term “short 1.5 GI promoter” refers to SEQ ID NO:28. The terms “short 1.6 GI promoter” and “short wild-type promoter” are used interchangeably and refer to SEQ ID NO:32. [0121]
  • The terms “glycerol-3-phosphate dehydrogenase” and “G3PDH” refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH; NADPH; or FAD-dependent. When specifically referring to a cofactor specific glycerol-3-phosphate dehydrogenase, the terms “NADH-dependent glycerol-3-phosphate dehydrogenase”, “NADPH-dependent glycerol-3-phosphate dehydrogenase” and “FAD-dependent glycerol-3-phosphate dehydrogenase” will be used. As it is generally the case that NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenases are able to use NADH and NADPH interchangeably (for example by the gene encoded by gpsA), the terms NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenase will be used interchangeably. The NADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by several genes including GPD1 (GenBank Z74071×2), or GPD2 (GenBank Z35169×1), or GPD3 (GenBank G984182), or DAR1 (GenBank Z74071×2). The NADPH-dependent enzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and L45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (GenBank Z47047×23), or glpD (GenBank G147838), or gipABC (GenBank M20938) (see WO 9928480 and references therein, which are herein incorporated by reference). [0122]
  • The terms “glycerol-3-phosphatase”, “sn-glycerol-3-phosphatase”, or “d,l-glycerol phosphatase”, and “G3P phosphatase” refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol-3-phosphate and water to glycerol and inorganic phosphate. G3P phosphatase is encoded, for example, by GPP1 (GenBank Z47047×125), or GPP2 (GenBank U18813x11) (see WO 9928480 and references therein, which are herein incorporated by reference). [0123]
  • The term “glycerol kinase” refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP. The high-energy phosphate donor ATP may be replaced by physiological substitutes (e.g., phosphoenolpyruvate). Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583×19) and glpK (GenBank L 19201) (see WO 9928480 and references therein, which are herein incorporated by reference). [0124]
  • The term “glycerol dehydrogenase” refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C. 1.1.1.72). A polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone is also referred to as a “dihydroxyacetone reductase”. Glycerol dehydrogenase may be dependent upon NADH (E.C. 1.1.1.6), NADPH (E.C. 1.1.1.72), or other cofactors (e.g., E.C. 1.1.99.22). A NADH-dependent glycerol dehydrogenase is encoded, for example, by gidA (GenBank U00006) (see WO 9928480 and references therein, which are herein incorporated by reference). [0125]
  • The term “dehydratase enzyme” or “dehydratase” will refer to any enzyme activity that catalyzes the conversion of a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention the dehydratase enzymes include a glycerol dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28) having preferred substrates of glycerol and 1,2-propanediol, respectively. Genes for dehydratase enzymes have been identified in [0126] Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, and Klebsiella oxytoca. In each case, the dehydratase is composed of three subunits: the large or “ ” subunit, the medium or “β” subunit, and the small or “γ” subunit. Due to the wide variation in gene nomenclature used in the literature, a comparative chart is given in Table 1 to facilitate identification. The genes are also described in, for example, Daniel et al. (FEMS Microbiol. Rev. 22, 553 (1999)) and Toraya and Mori (J. Biol. Chem. 274, 3372 (1999)). Referring to Table 1, genes encoding the large or “α” subunit of glycerol dehydratase include dhaB1, gldA and dhaB; genes encoding the medium or “β” subunit include dhaB2, gldB, and dhaC; genes encoding the small or “γ” subunit include dhaB3, gldC, and dhaE. Also referring to Table 1, genes encoding the large or “α” subunit of diol dehydratase include pduC and pddA; genes encoding the medium or “β” subunit include pduD and pddB; genes encoding the small or “γ” subunit include pduE and pddC.
    TABLE 1
    Comparative chart of gene names and GenBank references for dehydratase and dehydratase linked functions.
    GENE FUNCTION:
    1,3-PD
    ORGANISM regulatory unknown reactivation dehydrogenase unknown
    (GenBank base base base base base
    Reference) gene pairs gene pairs Geme pairs gene pairs gene pairs
    K. pneumoniae dhaR 2209-4134 orfW 4112-4642 OrfX 4643-4996 dhaT 5017-6108 orfY 6202-6630
    (SEQ ID NO: 1)
    K. pneumoniae orf2c 7116-7646 orf2b 6762-7115 dhaT 5578-6741 orf2a 5125-5556
    (U30903)
    K. pneumoniae GdrB
    (U60992)
    C. freundii dhaR 3746-5671 orfW 5649-6179 OrfX 6180-6533 dhaT 6550-7713 orfY 7736-8164
    (U09771)
    C. pasteurianum
    (AF051373)
    C. pasteurianum orfW 210-731 OrfX  1-196 dhaT 1232-2389 orfY  746-1177
    (AF006034)
    S. typhimurium PduH 8274-8645
    (AF026270)
    K. oxytoca DdrB 2063-2440
    (AF017781)
    K. oxytoca
    (AF051373)
    GENE FUNCTION:
    ORGANISM dehydratase, α dehydratase, β dehydratase, γ reactivation
    (GenBank base base base base
    Reference) gene pairs gene pairs Gene pairs gene pairs
    K. pneumoniae dhaB1 7044-8711 dhaB2 8724-9308 dhaB3 9311-9736 orfZ  9749-11572
    (SEQ ID NO:1)
    K. pneumoniae dhaB1 3047-4714 dhaB2 2450-2890 dhaB3 2022-2447 dhaB4  186-2009
    (U30903)
    K. pneumoniae gldA  121-1788 gldB 1801-2385 GldC 2388-2813 gdrA
    (U60992)
    C. freundii dhaB  8556-10223 dhaC 10235-10819 DhaE 10822-11250 orfZ 11261-13072
    (U09771)
    C. pasteurianum dhaB  84-1748 dhaC 1779-2318 DhaE 2333-2773 orfZ 2790-4598
    (AF051373)
    C. pasteurianum
    (AF006034)
    S. typhimurium pduC 3557-5221 pduD 5232-5906 PduE 5921-6442 pduG 6452-8284
    (AF026270)
    K. oxytoca ddrA  241-2073
    (AF017781)
    K. oxytoca pddA  121-1785 pddB 1796-2470 PddC 2485-3006
    (AF051373)
  • Glycerol and diol dehydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., [0127] FEMS Microbiol. Rev. 22, 553 (1999)). The term “dehydratase reactivation factor” refers to those proteins responsible for reactivating the dehydratase activity. The terms “dehydratase reactivating activity”, “reactivating the dehydratase activity” or “regenerating the dehydratase activity” refers to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved as the dehydratase reactivation factor (see WO 9821341 (US 6013494) and references therein, which are herein incorporated by reference; Daniel et al., supra; Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999)). Referring to Table 1, genes encoding one of the proteins include orfZ, dhaB4, gdrA, pduG and ddrA. Also referring to Table 1, genes encoding the second of the two proteins include orfX, orf2b, gdrB, pduH and ddrB.
  • The terms “1,3-propanediol oxidoreductase”, “1,3-propanediol dehydrogenase” or “DhaT” refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyzing the interconversion of 3-HPA and 1,3-propanediol provided the gene(s) encoding such activity is found to be physically or transcriptionally linked to a dehydratase enzyme in its natural (i.e., wild type) setting; for example, the gene is found within a dha regulon as is the case with dhaT from Klebsiella pneumoniae. Referring to Table 1, genes encoding a 1,3-propanediol oxidoreductase include dhaT from [0128] Klebsiella pneumoniae, Citrobacter freundii, and Clostridium pasteurianum. Each of these genes encode a polypeptide belonging to the family of type III alcohol dehydrogenases, exhibits a conserved iron-binding motif, and has a preference for the NAD+/NADH linked interconversion of 3-HPA and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050 (1987); Daniel et al., J. Bacteriol. 177, 2151 (1995); and Leurs et al., FEMS Microbiol. Lett. 154, 337 (1997)). Enzymes with similar physical properties have been isolated from Lactobacillus brevis and Lactobacillus buchneri (Veiga da Dunha and Foster, Appl. Environ. Microbiol. 58, 2005 (1992)).
  • The term “dha regulon” refers to a set of associated genes or open reading frames encoding various biological activities, including but not limited to a dehydratase activity, a reactivation activity, and a 1,3-propanediol oxidoreductase. Typically a dha regulon comprises the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ as described herein. [0129]
  • The term “non-specific catalytic activity” refers to the polypeptide(s) responsible for an enzyme activity that is sufficient to catalyze the interconversion of 3-HPA and 1,3-propanediol and specifically excludes 1,3-propanediol oxidoreductase(s). Typically these enzymes are alcohol dehydrogenases. Such enzymes may utilize cofactors other than NAD[0130] +/NADH, including but not limited to flavins such as FAD or FMN. A gene for a non-specific alcohol dehydrogenase (yqhD) is found, for example, to be endogenously encoded and functionally expressed within E. coli K12 strains.
  • The terms “function” or “enzyme function” refer to the catalytic activity of an enzyme in altering the energy required to perform a specific chemical reaction. It is understood that such an activity may apply to a reaction in equilibrium where the production of either product or substrate may be accomplished under suitable conditions. [0131]
  • The terms “polypeptide” and “protein” are used interchangeably. [0132]
  • The terms “carbon substrate” and “carbon source” refer to a carbon source capable of being metabolized by host microorganisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof. [0133]
  • The terms “host cell” or “host microorganism” refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product. [0134]
  • The terms “foreign gene”, “foreign DNA”, “heterologous gene” and “heterologous DNA” refer to genetic material native to one organism that has been placed within a host microorganism by various means. The gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene. [0135]
  • The terms “transformation” and “transfection” refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term “transformant” refers to the product of a transformation. [0136]
  • The term “genetically altered” refers to the process of changing hereditary material by transformation or mutation. [0137]
  • The terms “recombinant microorganism” and “transformed host” refer to any microorganism having been transformed with heterologous or foreign genes or extra copies of homologous genes. The recombinant microorganisms of the present invention express foreign genes encoding glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2 and dhaB3), dehydratase reactivation factor (orfZ and orfX), and optionally 1,3-propanediol oxidoreductase (dhaT) for the production of 1,3-propanediol from suitable carbon substrates. A preferred embodiment is an [0138] E. coli transformed with these genes but lacking a functional dhaT. A host microorganism, other than E. coli, may also be transformed to contain the disclosed genes and the gene for the non-specific catalytic activity for the interconversion of 3-HPA and 1,3-propanediol, specifically excluding 1,3-propanediol oxidoreductase(s) (dhaT).
  • “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding) and following (3′ non-coding) the coding region. The terms “native” and “wild-type” refer to a gene as found in nature with its own regulatory sequences. [0139]
  • The terms “encoding” and “coding” refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. It is understood that the process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. [0140]
  • The term “isolated” refers to a protein or DNA sequence that is removed from at least one component with which it is naturally associated. [0141]
  • An “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. [0142]
  • “Substantially similar” refers to nucleic acid molecules wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid molecules wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid molecule to mediate alteration of gene expression by antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid molecules of the instant invention (such as deletion or insertion of one or more nucleotide bases) that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. The invention encompasses more than the specific exemplary sequences. [0143]
  • For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups: [0144]
  • 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); [0145]
  • 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; [0146]
  • 3. Polar, positively charged residues: His, Arg, Lys; [0147]
  • 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and [0148]
  • 5. Large aromatic residues: Phe, Tyr, Trp. [0149]
  • Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product. [0150]
  • In many cases, nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. [0151]
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein. [0152]
  • A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. [0153] Molecular Cloning: A Laboratorv Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SSC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridization decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferable a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
  • A “substantial portion” refers to an amino acid or nucleotide sequence which comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., [0154] J. Mol. Biol. 215:403-410 (1993); see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid molecule comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid molecule comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for the purpose known to those skilled in the art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • The term “complementary” describes the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid molecules that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences. [0155]
  • The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: [0156] Computational Molecular Biology, Lesk, A. M., Ed.; Oxford University Press: New York, 1988; Biocomputing: Informatics and Genome Projects; Smith, D. W., Ed.; Academic Press: New York, 1993; Computer Analysis of Sequence Data, Part I; Griffin, A. M. and Griffin, H. G., Eds.; Humana Press: New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Ed.; Academic Press: New York, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds.; Stockton Press: New York, 1991. Preferred methods to determine identity are designed to give the largest match between the sequences tested.
  • Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG Pileup program found in the GCG program package, using the Needleman and Wunsch algorithm with their standard default values of gap creation penalty=12 and gap extension penalty=4 (Devereux et al., [0157] Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403410 (1990); Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402 (1997)). Another preferred method to determine percent identity, is by the method of DNASTAR protein alignment protocol using the Jotun-Hein algorithm (Hein et al., Methods Enzymol. 183:626-645 (1990)). Default parameters for the Jotun-Hein method for alignments are: for multiple alignments, gap penalty=11, gap length penalty=3; for pairwise alignments ktuple=6. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • The term “homologous” refers to a protein or polypeptide native or naturally occurring in a given host cell. The invention includes microorganisms producing homologous proteins via recombinant DNA technology. [0158]
  • The term “percent homology” refers to the extent of amino acid sequence identity between ppolypeptides. When a first amino acid sequence is identical to a ssecond amino acid sequence, then the first and second amino acid sequences exhibit 100% homology. The homology between any two polypeptides is a direct function of the total number of matching amino acids at a given position in either sequence, e.g., if half of the total number of amino acids in either of the two sequences are the same then the two sequences are said to exhibit 50% homology. [0159]
  • “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. [0160]
  • Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alteration in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. Moreover, the skilled artisan recognizes that sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequences exemplified herein. [0161]
  • The term “expression” refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product. [0162]
  • The terms “plasmid”, “vector”, and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host. [0163]
  • Construction of Recombinant Organisms [0164]
  • Recombinant organisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a carbon substrate to 1,3-propanediol may be constructed using techniques well known in the art. Genes encoding glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2, and dhaB3), dehydratase reactivation factor (orfZ and orfX) and 1,3-propanediol oxidoreductase (dhaT) were isolated from a native host such as Klebsiella or Saccharomyces and used to transform host strains such as [0165] E. coli DH5α, ECL707, M200, or KLP23.
  • Isolation of Genes [0166]
  • Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. [0167]
  • Alternatively, cosmid libraries may be created where large segments of genomic DNA (3545 kb) may be packaged into vectors and used to transform appropriate hosts. Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the foreign DNA. In addition to the cos sequence these vectors will also contain an origin of replication such as ColE1 and drug resistance markers such as a gene resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook, J. et al., supra. [0168]
  • Typically to clone cosmids, foreign DNA is isolated using the appropriate restriction endonucleases and ligated, adjacent to the cos region of the cosmid vector using the appropriate ligases. Cosmid vectors containing the linearized foreign DNA are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process the cos sites are cleaved and the foreign DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as [0169] E. coli. Once injected into the cell, the foreign DNA circularizes under the influence of the cos sticky ends. In this manner large segments of foreign DNA can be introduced and expressed in recombinant host cells.
  • Isolation and Cloning of Genes Encoding Glycerol Dehydratase (dhaB1, dhaB2, and dhaB3), Dehydratase Reactivating Factors (orfZ and orfX), and 1,3-propanediol dehydrogenase (dhaT) [0170]
  • Cosmid vectors and cosmid transformation methods were used within the context of the present invention to clone large segments of genomic DNA from bacterial genera known to possess genes capable of processing glycerol to 1,3-propanediol. Specifically, genomic DNA from [0171] K. pneumoniae was isolated by methods well known in the art and digested with the restriction enzyme Sau3A for insertion into a cosmid vector Supercos 1 and packaged using GigapackII packaging extracts. Following construction of the vector E. coli XL1-Blue MR cells were transformed with the cosmid DNA. Transformants were screened for the ability to convert glycerol to 1,3-propanediol by growing the cells in the presence of glycerol and analyzing the media for 1,3-propanediol formation.
  • Two of the 1,3-propanediol positive transformants were analyzed and the cosmids were named pKP1 and pKP2. DNA sequencing revealed extensive homology to the glycerol dehydratase gene from [0172] C. freundii, demonstrating that these transformants contained DNA encoding the glycerol dehydratase gene. Other 1,3-propanediol positive transformants were analyzed and the cosmids were named pKP4 and pKP5. DNA sequencing revealed that these cosmids carried DNA encoding a diol dehydratase gene.
  • Although the instant invention utilizes the isolated genes from within a Klebsiella cosmid, alternate sources of dehydratase genes and dehydratase reactivation factor genes include, but are not limited to, Citrobacter, Clostridia and Salmonella (see Table 1). [0173]
  • Genes Encoding G3PDH and G3P Phosphatase [0174]
  • The present invention provides genes suitable for the expression of G3PDH and G3P phosphatase activities in a host cell. [0175]
  • Genes encoding G3PDH are known. For example, GPD1 has been isolated from Saccharomyces (Wang et al., supra). Similarly, G3PDH activity has also been isolated from Saccharomyces encoded by GPD2 (Eriksson et al., [0176] Mol. Microbiol. 17, 95 (1995)).
  • For the purposes of the present invention it is contemplated that any gene encoding a polypeptide responsible for NADH-dependent G3PDH activity is suitable wherein that activity is capable of catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). Further, it is contemplated that any gene encoding the amino acid sequence of NADH-dependent G3PDH's corresponding to the genes DAR1, GPD1, GPD2, GPD3, and gpsA will be functional in the present invention wherein that amino acid sequence may encompass amino acid substitutions, deletions or additions that do not alter the function of the enzyme. The skilled person will appreciate that genes encoding G3PDH isolated from other sources will also be suitable for use in the present invention. Genes encoding G3P phosphatase are known. For example, GPP2 has been isolated from [0177] Saccharomyces cerevisiae (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)).
  • For the purposes of the present invention, any gene encoding a G3P phosphatase activity is suitable for use in the method wherein that activity is capable of catalyzing the conversion of glycerol-3-phosphate plus H[0178] 2O to glycerol plus inorganic phosphate. Further, any gene encoding the amino acid sequence of G3P phosphatase corresponding to the genes GPP2 and GPP1 will be functional in the present invention including any amino acid sequence that encompasses amino acid substitutions, deletions or additions that do not alter the function of the G3P phosphatase enzyme. The skilled person will appreciate that genes encoding G3P phosphatase isolated from other sources will also be suitable for use in the present invention.
  • Host Cells [0179]
  • Suitable host cells for the recombinant production of 1,3-propanediol may be either prokaryotic or eukaryotic and will be limited only by the host cell ability to express the active enzymes for the 1,3-propanediol pathway. Suitable host cells will be microorganisms such as Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. Preferred in the present invention are [0180] Escherichia coli, Escherichia blattae, Klebsiella, Citrobacter, and Aerobacter. Most preferred is E. coli (KLP23 (WO 2001012833 A2), RJ8.n (ATCC PTA4216), E. coli: FMP′::Km (ATCC PTA4732), MG 1655 (ATCC 700926)).
  • Vectors and Expression Cassettes [0181]
  • The present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor into a suitable host cell. Suitable vectors will be those which are compatible with the microorganism employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and using such vectors are known to those in the art (Sambrook et al., supra). [0182]
  • Initiation control regions, or promoters, which are useful to drive expression of the G3PDH and G3P phosphatase genes (DAR1 and GPP2, respectively) in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, λP[0183] L, λPR, T7, tac, and trc (useful for expression in E. coli).
  • Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. [0184]
  • For effective expression of the instant enzymes, DNA encoding the enzymes are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA. [0185]
  • Particularly useful in the present invention are the vectors pSYCO101, pSYCO103, pSYCO106, and pSYCO109. The essential elements are derived from the dha regulon isolated from [0186] Klebsiella pneumoniae and from Saccharomyces cerevlsiae. Each contains the open reading frames dhaB1, dhaB2, dhaB3, dhaX, orfX, DAR1, and GPP2 arranged in three separate operons, nucleotide sequences of which are given in SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively. The differences between the vectors are illustrated in the chart below [the prefix “p-” indicates a promoter; the open reading frames contained within each “( )” represent the composition of an operon]:
  • pSYCO101 (SEQ ID NO:65): [0187]
  • p-trc (Dar1_GPP2) in opposite orientation compared to the other 2 pathway operons, [0188]
  • p-1.6 long GI (dhaB1_dhaB2_dhaB3_dhaX), and [0189]
  • p-1.6 long GI (orfY_orfX_orfW). [0190]
  • pSYCO103 (SEQ ID NO:66): [0191]
  • p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, [0192]
  • p-1.5 long GI (dhaB1_dhaB2_dhaB3_dhaX), and [0193]
  • p-1.5 long GI (orfY_orfX_orfW). [0194]
  • pSYCO106 (SEQ ID NO:67): [0195]
  • p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, [0196]
  • p-1.6 long GI (dhaB1_dhaB2_dhaB3_dhaX), and [0197]
  • p-1.6 long GI (orfY_orfX_orfW). [0198]
  • pSYCO109 (SEQ ID NO:68): [0199]
  • p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, [0200]
  • p-1.6 long GI (dhaB1_dhaB2_dhaB3_dhaX), and [0201]
  • p-1.6 long GI (orfY_orfX). [0202]
  • Transformation of Suitable Hosts and Expression of Genes for the Production of 1,3-propanediol [0203]
  • Once suitable cassettes are constructed they are used to transform appropriate host cells. Introduction of the cassette containing the genes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor into the host cell may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, electroporation), or by transfection using a recombinant phage virus (Sambrook et al., supra). [0204]
  • In the present invention cassettes were used to transform the [0205] E. coli as fully described in the GENERAL METHODS and EXAMPLES.
  • Mutants [0206]
  • In addition to the cells exemplified, it is contemplated that the present method will be able to make use of cells having single or multiple mutations specifically designed to enhance the production of 1,3-propanediol. Cells that normally divert a carbon feed stock into non-productive pathways, or that exhibit significant catabolite repression could be mutated to avoid these phenotypic deficiencies. For example, many wild-type cells are subject to catabolite repression from glucose and by-products in the media and it is contemplated that mutant strains of these wild-type organisms, capable of 1,3-propanediol production that are resistant to glucose repression, would be particularly useful in the present invention. [0207]
  • Methods of creating mutants are common and well known in the art. For example, wild-type cells may be exposed to a variety of agents such as radiation or chemical mutagens and then screened for the desired phenotype. When creating mutations through radiation either ultraviolet (UV) or ionizing radiation may be used. Suitable short wave UV wavelengths for genetic mutations will fall within the range of 200 nm to 300 nm where 254 nm is preferred. UV radiation in this wavelength principally causes changes within nucleic acid sequence from guanidine and cytosine to adenine and thymidine. Since all cells have DNA repair mechanisms that would repair most UV induced mutations, agents such as caffeine and other inhibitors may be added to interrupt the repair process and maximize the number of effective mutations. Long wave UV mutations using light in the 300 nm to 400 nm range are also possible but are generally not as effective as the short wave UV light unless used in conjunction with various activators such as psoralen dyes that interact with the DNA. [0208]
  • Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect nonreplicating DNA such as HNO[0209] 2 and NH2OH, as well as agents that affect replicating DNA such as acridine dyes, notable for causing frameshift mutations. Specific methods for creating mutants using radiation or chemical agents are well documented in the art. See, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA., or Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992), herein incorporated by reference.
  • After mutagenesis has occurred, mutants having the desired phenotype may be selected by a variety of methods. Random screening is most common where the mutagenized cells are selected for the ability to produce the desired product or intermediate. Alternatively, selective isolation of mutants can be performed by growing a mutagenized population on selective media where only resistant colonies can develop. Methods of mutant selection are highly developed and well known in the art of industrial microbiology. See for example Brock, Supra; DeMancilha et al., [0210] Food Chem. 14, 313 (1984).
  • In addition to the methods for creating mutants described above, selected genes involved in converting carbon substrate to 1,3-propanediol may be up-regulated or down-regulated by a variety of methods which are known to those skilled in the art. It is well understood that up-regulation or down-regulation of a gene refers to an alteration in the activity of the protein encoded by that gene relative to a control level of activity, for example, by the activity of the protein encoded by the corresponding (or non-altered) wild-type gene. [0211]
  • Up-Regulation: [0212]
  • Specific genes involved in an enzyme pathway may be up-regulated to increase the activity of their encoded function(s). For example, additional copies of selected genes may be introduced into the host cell on multicopy plasmids such as pBR322. Such genes may also be integrated into the chromosome with appropriate regulatory sequences that result in increased activity of their encoded functions. The target genes may be modified so as to be under the control of non-native promoters or altered native promoters. Endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution. [0213]
  • Down-Regulation: [0214]
  • Alternatively, it may be useful to reduce or eliminate the expression of certain genes relative to a given activity level. For the purposes of this invention, it is useful to distinguish between reduction and elimination. The terms “down regulation” and “down-regulating” of a gene refers to a reduction, but not a total elimination, of the activity of the encoded protein. Methods of down-regulating and disrupting genes are known to those of skill in the art. [0215]
  • Down-regulation can occur by deletion, insertion, or alteration of coding regions and/or regulatory (promoter) regions. Specific down regulations may be obtained by random mutation followed by screening or selection, or, where the gene sequence is known, by direct intervention by molecular biology methods known to those skilled in the art. A particularly useful, but not exclusive, method to effect down-regulation is to alter promoter strength. [0216]
  • Disruption: [0217]
  • Disruptions of genes may occur, for example, by 1) deleting coding regions and/or regulatory (promoter) regions, 2) inserting exogenous nucleic acid sequences into coding regions and/regulatory (promoter) regions, and 3) altering coding regions and/or regulatory (promoter) regions (for example, by making DNA base pair changes). Such changes would either prevent expression of the protein of interest or result in the expression of a protein that is non-functional. Specific disruptions may be obtained by random mutation followed by screening or selection, or, in cases where the gene sequences in known, specific disruptions may be obtained by direct intervention using molecular biology methods know to those skilled in the art. A particularly useful method is the deletion of significant amounts of coding regions and/or regulatory (promoter) regions. [0218]
  • Methods of altering recombinant protein expression are known to those skilled in the art, and are discussed in part in Baneyx, [0219] Curr. Opinion Biotech. (1999) 10:411; Ross, et al., J. Bacteriol. (1998) 180:5375; deHaseth, et al., J. Bacteriol. (1998) 180:3019; Smolke and Keasling, Biotech. And Bioengineeering (2002) 80:762; Swartz, Curr. Opinions Biotech.(2001) 12:195; and Ma, et al., J. Bacteriol. (2002) 184:5733.
  • Alterations in the 1.3-propanediol Production Pathway [0220]
  • Representative Enzyme Pathway. The production of 1,3-propanediol from glucose can be accomplished by the following series of steps. This series is representative of a number of pathways known to those skilled in the art. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases, which are known to be non-specific with respect to their substrates, or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD[0221] + (or NADP+) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) that catalyzes the reversible reaction of Equation 3.
  • Glycerol→3-HPA+H2O  (Equation 1)
  • 3-HPA+NADH+H+→1,3-Propanediol+NAD+  (Equation 2)
  • Glycerol+NAD+→DHA+NADH+H+  (Equation 3)
  • Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxy-propionaldehye (3-HPA) as has been described in detail above. The intermediate 3-HPA is produced from glycerol, Equation 1, by a dehydratase enzyme that can be encoded by the host or can be introduced into the host by recombination. This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or any other enzyme able to catalyze this transformation. Glycerol dehydratase, but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediol is produced from 3-HPA, Equation 2, by a NAD[0222] +- (or NADP+) linked host enzyme or the activity can be introduced into the host by recombination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.
  • Mutations and transformations that affect carbon channeling. A variety of mutant microorganisms comprising variations in the 1,3-propanediol production pathway will be useful in the present invention. Mutations which block alternate pathways for intermediates of the 1,3-propanediol production pathway would also be useful to the present invention. For example, the elimination of glycerol kinase prevents glycerol, formed from G3P by the action of G3P phosphatase, from being re-converted to G3P at the expense of ATP. Also, the elimination of glycerol dehydrogenase (for example, gidA) prevents glycerol, formed from DHAP by the action of NADH-dependent glycerol-3-phosphate dehydrogenase, from being converted to dihydroxyacetone. Mutations can be directed toward a structural gene so as to impair or improve the activity of an enzymatic activity or can be directed toward a regulatory gene, including promoter regions and ribosome binding sites, so as to modulate the expression level of an enzymatic activity. [0223]
  • It is thus contemplated that transformations and mutations can be combined so as to control particular enzyme activities for the enhancement of 1,3-propanediol production. Thus, it is within the scope of the present invention to anticipate modifications of a whole cell catalyst which lead to an increased production of 1,3-propanediol. [0224]
  • The present invention utilizes a preferred pathway for the production of 1,3-propanediol from a sugar substrate where the carbon flow moves from glucose to DHAP, G3P, Glycerol, 3-HPA, and finally to 1,3-propanediol. The present production strains have been engineered to maximize the metabolic efficiency of the pathway by incorporating various deletion mutations that prevent the diversion of carbon to non-productive compounds. Glycerol may be diverted from conversion to 3HPA by transformation to either DHA or G3P via glycerol dehydrogenase or glycerol kinase as discussed above. Accordingly, the present production strains contain deletion mutations in the gidA and glpk genes. Similarly DHAP may be diverted to 3-PG by triosephosphate isomerase, thus the present production microorganism also contains a deletion mutation in this gene. The present method additionally incorporates a dehydratase enzyme for the conversion of glycerol to 3HPA, which functions in concert with the reactivation factor, encoded by orfX and orfZ of the dha regulon. Although conversion of the 3HPA to 1,3-propanediol is typically accomplished via a 1,3-propanediol oxidoreductase, the present method utilizes a non-specific catalytic activity that produces greater titers and yields of the final product, 1,3-propanediol. In such a process, titers of 1,3-propanediol of at least 10 g/L are achieved, where titers of 200 g/L are expected. [0225]
  • Alternatively, an improved process for 1,3-propanediol production may utilize glycerol or dihydroxyacetone as a substrate where the pathway comprises only the last three substrates, glycerol→3HPA→1,3-propanediol. In such a process, the oxidoreductase is again eliminated in favor of the non-specific catalytic activity (expected to be an alcohol dehydrogenase), however the need for deletion mutations are nullified by the energy considerations of adding glycerol to the culture. In such as process titers of 1,3-propanediol of at least 71 g/L are achieved where titers of 200 g/L are expected. [0226]
  • Similarly it is within the scope of the invention to provide mutants of wild-type microorganisms that have been modified by the deletion or mutation of the dhaT activity to create improved 1,3-propandiol producers. For example, microorganisms, which naturally contain all the elements of the dha regulon, may be manipulated so as to inactivate the dhaT gene encoding the 1,3-propanediol oxidoreductase activity. These microorganisms will be expected to produce higher yields and titers of 1,3-propanediol, mediated by the presence of an endogenous catalytic activity, expected to be an alcohol dehydrogenase. Examples of such microorganisms include but are not limited to Klebsiella sp., Citrobacter sp., and Clostridium sp. [0227]
  • Media and Carbon Substrates [0228]
  • Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose and oligosaccharides such as lactose or sucrose. [0229]
  • In the present invention, the preferred carbon substrate is glucose. [0230]
  • In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 1,3-propanediol production. Particular attention is given to Co(II) salts and/or vitamin B[0231] 12 or precursors thereof.
  • Adenosyl-cobalamin (coenzyme B[0232] 12) is an essential cofactor for dehydratase activity. Synthesis of coenzyme B12 is found in prokaryotes, some of which are able to synthesize the compound de novo, for example, Escherichia blattae, Klebsiella species, Citrobacter species, and Clostridium species, while others can perform partial reactions. E. coli, for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to corrinoid and can introduce the 5′-deoxyadenosyl group. Thus, it is known in the art that a coenzyme B12 precursor, such as vitamin B12, need be provided in E. coli fermentations.
  • Vitamin B[0233] 12 additions to E. coli fermentations may be added continuously, at a constant rate or staged as to coincide with the generation of cell mass, or may be added in single or multiple bolus additions. Preferred ratios of vitamin B12 (mg) fed to cell mass (OD550) are from 0.06 to 0.60. Most preferred ratios of vitamin B12 (mg) fed to cell mass (OD550) are from 0.12 to 0.48.
  • Although vitamin B[0234] 12 is added to the transformed E. coli of the present invention it is contemplated that other microorganisms, capable of de novo B12 biosynthesis will also be suitable production cells and the addition of B12 to these microorganisms will be unnecessary.
  • Culture Conditions: [0235]
  • Typically cells are grown at 35° C. in appropriate media. Preferred growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by someone skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the reaction media. Similarly, the use of agents known to modulate enzymatic activities (e.g., methyl viologen) that lead to enhancement of 1,3-propanediol production may be used in conjunction with or as an alternative to genetic manipulations. [0236]
  • Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition. [0237]
  • Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism. [0238]
  • Fed-batch fermentations may be performed with carbon feed, for example, glucose, limited or excess. [0239]
  • Batch and Continuous Fermentations: [0240]
  • The present process employs a batch method of fermentation. Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the media is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur adding nothing to the system. Typically, however, “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate. [0241]
  • A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO[0242] 2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, supra.
  • Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. [0243]
  • Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra. [0244]
  • It is contemplated that the present invention may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 1,3-propanediol production. [0245]
  • Identification and Purification of 1.3-propanediol: [0246]
  • Methods for the purification of 1,3-propanediol from fermentation media are known in the art. For example, propanediols can be obtained from cell media by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. Pat. No. 5,356,812). A particularly good organic solvent for this process is cyclohexane (U.S. Pat. No. 5,008,473). [0247]
  • 1,3-Propanediol may be identified directly by submitting the media to high pressure liquid chromatography (HPLC) analysis. Preferred in the present invention is a method where fermentation media is analyzed on an analytical ion exchange column using a mobile phase of 0.01 N sulfuric acid in an isocratic fashion. [0248]
    • General Methods and Materials
  • Procedures for phosphorylations, ligations and transformations are well known in the art. Techniques suitable for use in the following examples may be found in Sambrook, J. et al., supra. [0249]
  • Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found in [0250] Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.
  • The meaning of abbreviations is as follows: “hr” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “mL” means milliliters, “L” means liters, 50 amp is 50 μg/mL ampicillin, and LB-50 amp is Luria-Bertani broth containing 50 μg/mL ampicillin. [0251]
  • Within the tables the following abbreviations are used. “Con.” is conversion, “Sel.” is selectivity based on carbon, and “nd” is not detected. [0252]
  • Strains and vectors used and constructed in the following examples are listed in the chart below: [0253]
    Name Alternate name Phenotype
    FM5
    RJF10 FM5 glpK-
    KLP23 FM5 glpK- gldA-
    WO2001012833 A2
    KLndh81 FM5 glpk- gldA- ndh-
    KLpts7 FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    KLgalP-trc FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc
    KLGG FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc glkp-trc
    KLGG ΔarcA FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc glkp-trc arcA-
    KLGG ΔarcA Δedd FMP FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc glkp-trc arcA- edd-
    Selected FMP FMP'::Km FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc glkp-trc* arcA- edd-
    FMP'::Km 1.5 gapA FM5 glpk- gldA- ndh- ptsHlcrr- KmR
    galPp-trc glkp-trc* arcA- edd- gapAp-1.5
    FMP' 1.5 gapA FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    glkp-trc* arcA- edd- gapAp-1.5
    FMP' 1.5 gapA ΔmgsA FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    FMP' 1.5 gap Δmgs Triple FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    1.6 ppc glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6
    Triple 1.6 btuR FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6
    Triple 1.6 btuR FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    1.6yqhD glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6 yqhC- yqhDp-1.6
    Triple 1.6 btuR Triple FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    1.6yqhD ΔackA-pta Triple glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6 yqhC- yqhDp-1.6
    ackA-pta-
    Triple Triple ΔaldA FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6 yqhC- yqhDp-1.6
    ackA-pta- aldA-
    Triple Triple ΔaldB FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6 yqhC- yqhDp-1.6
    ackA-pta- aldB-
    Triple Triple ΔaldA FM5 glpk- gldA- ndh- ptsHlcrr- galPp-trc
    ΔaldB glkp-trc* arcA- edd- gapAp-1.5 mgsA-
    ppcp-1.6 yciK-btuRp-1.6 yqhC- yqhDp-1.6
    ackA-pta- aldA- aldB-
  • Strains: [0254]
    KLP23 (WO 2001012833 A2),
    RJ8.n (ATCC PTA-4216),
    MG 1655 ATCC 700926 (commercially available)
  • Plasmids: [0255]
    pAH48 WO 9821340 A1
    pDT29 WO 2001012833 A2
    pKP32 WO 2001012833 A2
    pSYCO101 SEQ ID NO: 65.
    pSYCO103 SEQ ID NO: 66.
    pSYCO106 SEQ ID NO: 67.
    pSYCO109 SEQ ID NO: 68.
  • The plasmids pKD3, pKD4, pKD13 and pKD46, and pCP20 have been described (Datsenko and Wanner, supra). The plasmids pLoxCat2 and pJW168 have been described (Palmeros et al., supra). [0256]
  • Chromosomal Integration for Gene Knockouts, Promoter Replacements and Introduction of Chromosomal Mutations. [0257]
  • To integrate DNA into a specific region of the chromosome, homology of the inserting DNA to the targeted chromosomal site and a selectable marker are required. It is advantageous if the marker can be easily removed after integration. The loxP/Cre recombinase system from P1 phage and the FRT/Flp recombinase system from yeast provide a mechanism to remove the marker. The loxP and FRT sites are recognition sites for the Cre and Flp recombinases. Cre and Flp are site specific recombinases, which excise the intervening DNA from the directly repeated recognition sites. [0258]
  • The integration cassette containing homologous arms to the targeted chromosomal site and encoding a selectable marker flanked by loxP [Palmeros et al. [0259] Gene 247, 255-264 (2000)] or FRT [Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97, 6640-6645 (2000)] sites is transformed into target cells harboring pKD46 (Datsenko and Wanner, supra). Successful integrants are selected by growth of the cells in the presence of the antibiotic. Subsequently, pKD46 is cured from the cells and the recombinase plasmids are then introduced into the integrants for removal of the antibiotic gene. Strains integrated with a loxP cassette are transformed with pJW168 that encodes Cre recombinase (Palmeros et al, supra). Strains containing a FRT cassette are transformed with pCP20 that encodes Fip recombinase (Datsenko and Wanner, supra). After removal of the integrated marker, the recombinase plasmids are cured from the strain.
  • P1 virtransduction were performed as previously described [Miller, J. H., A short course in bacterial genetics. A laboratory manual and handbook for [0260] Escherchia coli and related bacteria (1992), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.].
  • Enzyme Assays [0261]
  • Assays for Glucokinase (Glk) Activity: [0262]
  • Glucokinase (Glk) activity was assayed by following the conversion of glucose to glucose-6-phosphate spectrophotometrically at 340 nm by coupling the glucokinase reaction with that of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The assay contained 0.5 mM NADP, 5 mM ATP, 5 mM MgCl, and 2 units of glucose-6-phosphate dehydrogenase in 100 mM phosphate buffer, pH 7.2. Alternative assays may be found in T. E. Barman, Enzyme Handbook (1985), Springer-Verlag, Berlin. [0263]
  • Assay for Glyceraldehyde-3-Phosphate Dehydrogenase Activity: [0264]
  • Assay for Glyceraldehyde-3-phosphate dehydrogenase activity was measured in cell-free extracts by the appearance of NADH. An ultracentrifuged (50,000×g, 1 h, 4° C.) cell-free supernatant was partially purified using an ion exchange column prior to assay. The assay contained 0.2 mM glyceraldehyde 3-phosphate, 2.5 mM NAD[0265] +, 2 mM EDTA, 5 mM cysteamine, 20 mM potassium phosphate and 40 mM triethanolamine at pH 8.9. Alternative assays may be found in T. E. Barman, supra.
  • Assay for Phosphoenolpyruvate Carboxylase (Ppc) Activity: [0266]
  • Phosphoenolpyruvate carboxylase (Ppc) activity was measured in cell-free extracts by a coupled assay (Flores and Gancedo, [0267] FEBS Lett. 412, 531-534 (1997)). This method involved incubating at room temperature a ultracentifuged (50,000×g, 1 h, 4° C.) cell-free extract sample in a cuvette that contained 0.22 mM NADH, 1.1 mM phosphoenolpyruvate (PEP), 0.25 mM acetyl-CoA, and 6 U of malate dehydrogenase (MDH) in 0.1 M Tris/HCl buffer, pH 8.5, with 11 mM sodium bicarbonate and 11 mM magnesium sulfate, in a total volume of 1.0 mL. A background rate of the reaction of enzyme and NADH was first determined at 340 nm in the absence of PEP. The second substrate, PEP, was subsequently added and the absorbance change over time was further monitored. Ppc activity was defined by subtracting the background rate from the gross rate. Alternative assays may be found in T. E. Barman, supra.
  • Assays for Dehydratase Enzymes: [0268]
  • Dehydratase activity in cell-free extracts was determined using either glycerol or 1,2-propanediol as substrate. Typically, cell-free extracts were prepared by cell disruption using a french press followed by centrifugation of the cellular debris. The assay, based on the reaction of aldehydes with methylbenzo-2-thiazolone hydrazone, has been described by Forage and Foster ([0269] Biochim. Biophys. Acta 569, 249 (1979)).
  • Honda et al. ([0270] J. Bacteriol. 143, 1458 (1980)) disclose an assay that measures the reactivation of dehydratases. Dehydratase activity was determined in toluenized whole cells, with and without ATP, using either glycerol or 1,2-propanediol as substrate. Reactivation was determined by the ratio of product formation with versus without the ATP addition. Product formation (3-HPA or propionaldehyde when glycerol or 1,2-propanediol is used as substrate, respectively) was measured directly, using HPLC, or indirectly, using the methylbenzo-2-thiazolone hydrazone reagent. Alternatively, product formation was determined by coupling the conversion of the aldehyde to its respective alcohol using a NADH linked alcohol dehydrogenase and monitoring the disappearance of NADH.
  • Assays for 1,3-propanediol Oxidoreductase: [0271]
  • The activity of 1,3-propanediol oxidoreductase, sometimes referred to as 1,3-propanediol dehydrogenase, was determined for cell-free extracts in solution or in slab gels using 1,3-propanediol and NAD[0272] + as substrates has been described (Johnson and Lin, J. Bacteriol. 169, 2050 (1987)). Alternatively, the conversion of 3-HPA and NADH to 1,3-propanediol and NAD+ was determined by the disappearance of NADH. The slab gel assay has the potential advantage of separating the activity of 1,3-propanediol oxidoreductase (dhaT) from that of non-specific alcohol dehydrogenases by virtue of size separation. The native molecular weights of 1,3-propanediol oxidoreductases (dhaT) from Citrobacter frendii, Klebsiella pneumoniae, and Clostridium pasteurianum are unusually large, on the order of 330,000 to 440,000 daltons. Lactobacillus brevis and Lactobacillus buchneri contain dehydratase associated 1,3-propanediol oxidoreductases with properties similar to those of known 1,3-propanediol oxidoreductases (dhaT).
  • Assays for Glycerol 3-phosphate Dehydrogenase Activity: [0273]
  • A procedure was used as modified below from a method published by Bell et al. ([0274] J. Biol. Chem. 250, 7153 (1975)). This method involved incubating a cell-free extract sample in a cuvette that contained 0.2 mM NADH, 2.0 mM dihydroxyacetone phosphate (DHAP), and enzyme in 0.1 M Tris/HCl, pH 7.5 buffer with 5 mM DTT, in a total volume of 1.0 mL at 30° C. A background rate of the reaction of enzyme and NADH was first determined at 340 nm for at least 3 min. The second substrate, DHAP, was subsequently added and the absorbance change over time was further monitored for at least 3 min. G3PDH activity was defined by subtracting the background rate from the gross rate.
  • Assay for Glycerol-3-phosphatase Activity: [0275]
  • The assay for enzyme activity was performed by incubating the extract with an organic phosphate substrate in a bis-Tris or MES and magnesium buffer, pH 6.5. The substrate used was either I-α-glycerol phosphate, or d,l-α-glycerol phosphate. The final concentrations of the reagents in the assay are: buffer (20 mM, bis-Tris or 50 mM MES); MgCl[0276] 2 (10 mM); and substrate (20 mM). If the total protein in the sample was low and no visible precipitation occurs with an acid quench, the sample was conveniently assayed in the cuvette. This method involved incubating an enzyme sample in a cuvette that contained 20 mM substrate (50 μL, 200 mM), 50 mM MES, 10 mM MgCl2, pH 6.5 buffer. The final phosphatase assay volume was 0.5 mL. The enzyme-containing sample was added to the reaction mixture; the contents of the cuvette were mixed and then the cuvette was placed in a circulating water bath at T=37° C. for 5 to 120 min, the length of time depending on whether the phosphatase activity in the enzyme sample ranged from 2 to 0.02 U/mL. The enzymatic reaction was quenched by the addition of the acid molybdate reagent (0.4 mL). After the Fiske SubbaRow reagent (0.1 mL) and distilled water (1.5 mL) were added, the solution was mixed and allowed to develop. After 10 min, to allow full color development, the absorbance of the samples was read at 660 nm using a Cary 219 UV/vis spectrophotometer. The amount of inorganic phosphate released was compared to a standard curve that was prepared by using a stock inorganic phosphate solution (0.65 mM) and preparing 6 standards with final inorganic phosphate concentrations ranging from 0.026 to 0.130 μmol/mL.
  • Assay for Glycerol Kinase Activity: [0277]
  • An appropriate amount of enzyme, typically a cell-free crude extract, was added to a reaction mixture containing 40 mM ATP, 20 mM MgSO[0278] 4, 21 mM uniformly 13C labeled glycerol (99%, Cambridge Isotope Laboratories), and 0.1 M Tris-HCl, pH 9 for 75 min at 25° C. The conversion of glycerol to glycerol 3-phosphate was detected by 13C-NMR (125 MHz): glycerol (63.11 ppm, 6, J=41 Hz and 72.66 ppm, t, J=41 Hz); glycerol 3-phosphate (62.93 ppm, 6, J=41 Hz; 65.31 ppm, br d, J=43 Hz; and 72.66 ppm, dt, J=6, 41 Hz).
  • NADH-Linked Glycerol Dehydrogenase Assay: [0279]
  • NADH-linked glycerol dehydrogenase activity (gidA) in cell-free extracts from [0280] E. coli strains was determined after protein separation by non-denaturing polyacrylamide gel electrophoresis. The conversion of glycerol plus NAD+ to dihydroxyacetone plus NADH was coupled with the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to a deeply colored formazan, using phenazine methosulfate (PMS) as mediator (Tang et al., J. Bacteriol. 140, 182 (1997)).
  • Electrophoresis was performed in duplicate by standard procedures using native gels (8-16% TG, 1.5 mm, 15 lane gels from Novex, San Diego, Calif.). Residual glycerol was removed from the gels by washing 3× with 50 mM Tris or potassium carbonate buffer, pH 9 for 10 min. The duplicate gels were developed, with and without glycerol (approximately 0.16 M final concentration), in 15 mL of assay solution containing 50 mM Tris or potassium carbonate, [0281] pH 9, 60 mg ammonium sulfate, 75 mg NAD+, 1.5 mg MTT, and 0.5 mg PMS.
  • The presence or absence of NADH-linked glycerol dehydrogenase activity in [0282] E. coli strains (gidA) was also determined, following polyacrylamide gel electrophoresis, by reaction with polyclonal antibodies raised to purified K. pneumoniae glycerol dehydrogenase (dhaD).
  • Isolation and Identification of 1,3-propanediol: [0283]
  • HPLC analysis of fermentation products. The conversion of glucose to 1,3-propanediol was monitored by HPLC. Analyses were performed using standard chromatography. One suitable method utilized a Waters Alliance HPLC system using R1 detection. Samples were injected onto a Aminex HPX87H column (7.8 mm×300 mm, Biorad, Hercules, Calif.) equipped with a Cation H Refill Cartridge precolumn (4.6 mm×30 mm, Biorad, Hercules, Calif.), temperature controlled at 50° C., using 5 mM H[0284] 2SO4 as mobile phase at a flow rate of 0.4 mL/min. The system was calibrated weekly against standards of known concentration. Typically, the retention times of glucose, glycerol, 1,3-propanediol, and acetic acid were 12.7 min, 19.0 min, 25.2 min, and 21.5 min, respectively.
  • GC/MS Analysis of Fermentation Methods. [0285]
  • Production of 1,3-propanediol was confirmed by GC/MS. Analyses were performed using standard techniques and materials available to one of skill in the art of GC/MS. One suitable method utilized a Hewlett Packard 5890 Series II gas chromatograph coupled to a Hewlett Packard 5971 Series mass selective detector (EI) and a HP-INNOWax column (30 m length, 0.25 mm i.d., 0.25 micron film thickness). The retention time and mass spectrum of 1,3-propanediol generated were compared to that of authentic 1,3-propanediol (rn/e: 57, 58). [0286]
  • An alternative method for GC/MS involved derivatization of the sample. To 1.0 mL of sample (e.g., culture supernatant) was added 30 μL of concentrated (70% v/v) perchloric acid. After mixing, the sample was frozen and lyophilized. A 1:1 mixture of bis(trimethylsilyl)trifluoroacetamide:pyridine (300 μL) was added to the lyophilized material, mixed vigorously and placed at 65° C. for one h. The sample was clarified of insoluble material by centrifugation. The resulting liquid partitioned into two phases, the upper of which was used for analysis. The sample was chromatographed on a DB-5 column (48 m, 0.25 mm I.D., 0.25 μm film thickness; from J&W Scientific) and the retention time and mass spectrum of the 1,3-propanediol derivative obtained from culture supernatants were compared to that obtained from authentic standards. The mass spectra of TMS-derivatized 1,3-propanediol contains the characteristic ions of 205, 177, 130 and 115 AMU. [0287]
  • Medium Composition [0288]
  • TM2 medium (TM2) is a base recipe to which carbon source (typically glucose, at 20 g/L or 40 g/L), appropriate antibiotics, and other components are added. TM2 medium contains the following components: K[0289] 2HPO4 (13.6 g/L), KH2PO4 (13.6 g/L), MgSO47H2O (2 g/L), citric acid monohydrate (2 g/L), ferric ammonium citrate (0.3 g/L), (NH4)2SO4 (3.2 g/L), yeast extract (5 g/L), solution of trace elements (1 ml). The pH is adjusted to 6.8. The solution of trace elements contains: citric acid H2O (4.0 g/L), MnSO4.H2O (3.0 g/L), NaCl (1.0 g/L), FeSO4.7H2O (0.10 g/L), COCl2.6H2O (0.10 g/L), ZnSO4.7H2O (0.10 g/L), CuSO4.5H2O (0.010 g/L), H3BO3 (0.010 g/L), and Na2MoO4.2H2O (0.010 g/L).
  • TM3 medium (TM3) is identical to TM2 medium except that it contains 0.5 g/L yeast extract. [0290]
  • LB medium (LB) contains 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl. LB plates (or LA) are LB medium+2% agar. 2YT medium (2YT) contains 10 g/L yeast extract, 16 g/L tryptone, and 10 g/L NaCl. Soy broth with glucose (SBG1%) contains 10 g/L Soytone (Difco), 5 g/L yeast extract, 10 g/L NaCl, and 10 g/L glucose. [0291]
  • Fermentation Protocol (14L) [0292]
  • Shake flask cultures of all strains described (KLP23, RJ8.n, MB 1655) were grown on either 2YT medium or LB containing the appropriate antibiotics as detailed in the examples to make the pre-culture for inoculation of the fermenters. Cultures were started from either frozen seed vials prepared with 15% glycerol as a cryoprotectant or from a single colony grown on fresh LA plates with 50 mg/L spectinomycin. Cultures started with a frozen vial were grown in 500 mL of the specified medium in a 2L flask; when a single colony was used to start the pre-culture, it was placed in 30 mL of the specified medium in a 250 mL baffled flask. The cultures were incubated at 34° C. and 300 rpm shaking to an OD[0293] 550 of approximately 1.0 AU was reached and used to seed the fermenter. In some cases, a seed fermenter was used to provide a larger pre-culture for inoculating a production fermenter. Seed fermenters were generally identical to production fermenters except that vitamin B12 was not added to the seed tank. Details about the procedures for using seed fermenters are described in the pertinent examples.
  • Seed and production fermenters were prepared with the same medium, containing KH[0294] 2PO4 (7.5 g/L), MgSO4.7H2O (2 g/L), citric acid monohydrate (2 g/L), ferric ammonium citrate (0.3 g/L), CaCl2,2H2O (0.2 g/L), sulfuric acid (98%; 1.2 mL/L), Mazu DF204 (0.4 mL/L) as antifoam, yeast extract (5 g/L), solution of trace elements (10 ml/L). The solution of trace elements contains: citric acid H2O (4.0 g/L), MnSO4.H2O (3.0 g/L), NaCl (1.0 g/L), FeSO4.7H2O (0.10 g/L), COCl2.6H2O (0.10 g/L), ZnSO4. 7H2O (0.10 g/L), CuSO4.5H2O (0.010 g/L), H3BO3 (0.010 g/L), and Na2MoO4.2H2O (0.010 g/L). After sterilization, the pH was adjusted to 6.8 with 20-28% NH4OH and additions of glucose (to 10-25 g/L from a 60-67% (w/w) solution) and the appropriate antibiotics (see specific examples for details) were made. The fermenter volume after inoculation was 6 liters.
  • A 14 L stirred tank fermenter was prepared with the medium described above. The temperature was controlled at 34° C. and aqueous ammonia (20-28 weight %) was used to control pH at 6.8. Backpressure was controlled at 0.5 barg and dO control set at 5%. Except for minor excursions, glucose concentration was maintained between 0 g/L and 25 g/L with a 60-67% (w/w) feed. Vitamin B[0295] 12 additions and any other changes to the general procedure described here are noted in the examples.
  • Molar yield, as either fraction or %, represents (mol glycerol produced+mol 1,3-propanediol produced)/(mol glucose consumed). Weight yield, generally given as %, represents (g 1,3-propanediol produced)/(g glucose consumed). [0296]
    • EXAMPLE 1 Construction of NADH Dehydrogenase II minus (Δndh) E. coli Strains for the Production of 1,3-Propanediol
  • Construction of KLndh81. [0297]
  • An ndh mutation was obtained by interrupting the coding region with a loxP511 cassette. The ndh gene (for reference, see GenBank, Accession # U00096), with upstream and downstream flanking regions, was PCR amplified from [0298] E. coli MG1655 and cloned. The ndh cassette was digested with Stu I, cutting roughly in the middle of the gene, and a loxP511-Cat-loxP511 cassette was cloned into this site with the cat gene in the opposite orientation relative to the ndh gene. The loxP511-Cat-/oxP511 cassette was obtained from the plasmid pLoxCat27 [SEQ ID NO:1] by digestion with Spe I and EcoR V, followed by fill-in to generate blunt ends, and gel purification of the 1.1 kb fragment. The loxP511 site is a variant of the loxP site (Palmeros et al., supra). The ndh::Cat cassette was PCR amplified and electroporated into KLP23 competent cells creating strain KLndh81::Cm. The chloramphenicol marker was removed by the Cre recombinase (Palmeros et al., supra) leaving 96 bp of interrupting sequence containing one loxP511 site. This strain was designated KLndh81. Alternatively, an ndh mutation was obtained by interrupting the coding region with a Cat cassette without loxP sites to give strain KLNDH413.
  • Construction of RJ8.n. [0299]
  • A cassette containing ndh flanking sequence and loxP511-Cat-loxP511 from Klndh81::Cm was PCR amplified and cloned into pUni/V5-His TOPO [Invitrogen] to create pAH111. The ndh-loxP511-Cat-loxP511 cassette from pAH111 was integrated into strain RJ8/pKD46. Recombinant strains were selected for chloramphenicol resistance. Successful integration of the cassette into ndh was confirmed by PCR. The chloramphenicol marker was removed by using Cre recombinase (Palmeros et al., supra) creating strain RJ8.n. [0300]
    • EXAMPLE 2 Comparison of 1,3-Propanediol and Glycerol Production with E. coli Strains KLP23/pAH48/pDT29 and KLP23/pAH48/pKP32
  • Strain KLP23 was transformed with plasmids pAH48 and pDT29 or pKP32. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods. Pre-cultures for each fermentation were prepared using frozen vials thawed and grown in 500 mL 2YT with 200 mg/L carbenicillin and 50 mg/L spectinomycin. The full contents of the flask were used to inoculate the fermenter. The fermenter was operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods. The vitamin B[0301] 12 strategy for each fermentation is detailed below.
  • Fermentation with KLP23/PAH48/pDT29. [0302]
  • In this example, vitamin B[0303] 12 (0.075 g/L, 500 mL) was fed, starting 3 h after inoculation, at a rate of 16 mL/h. A representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pDT29 is given in Table 2.1. The yield of 1,3-propanediol was 24 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 68 g/L 1,3-propanediol was obtained.
    TABLE 2.1
    Representative fermentation summary of the conversion
    of glucose to 1,3-propanediol (1,3-PD) using
    E. coli strain KLP23/pAH48/pDT29
    Time OD550 DO Glucose Glycerol 1,3-PD
    (h) (AU) (%) (g/L) (g/L) (g/L)
    0 0 150 12.9 0.0 0
    6 17 80 8.3 3.1 1
    12 42 53 2.8 12.5 9
    18 98 9 5.7 12.6 32
    24 136 11 32.8 12.0 51
    30 148 10 12.3 13.3 62
    32 152 11 12.5 14.3 65
    38 159 11 1.5 17.2 68
  • Similar results were obtained with an identical vitamin B[0304] 12 feed at twice the concentration or bolus additions of vitamin B12 across the time course of the fermentation. The highest titer obtained was 77 g/L.
  • Improved Fermentation with KLP23/pAH48/pKP32. [0305]
  • A representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using [0306] E. coli strain KLP23/pAH48/pKP32 is given in Table 2.2. Vitamin B12 (0.150 g/L, 500 mL) was fed, starting 3 h after inoculation, at a rate of 16 mL/h. After 36 h, approximately 2 L of fermentation broth was purged in order to allow for the continued addition of glucose feed. The yield of 1,3-propanediol was 26 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 112 g/L 1,3-propanediol was obtained.
    TABLE 2.2
    Representative fermentation summary of the improved
    conversion of glucose to 1,3-propanediol (1,3-PD)
    using E. coli strain KLP23/pAH48/pKP32
    Time OD550 DO Glucose Glycerol 1,3-PD
    (h) (AU) (%) (g/L) (g/L) (g/L)
    0 0 148 12.8 0.0 0
    6 22 84 6.9 3.3 0
    12 34 90 9.7 10.4 7
    18 66 43 9.3 5.9 24
    24 161 9 0.2 2.5 46
    30 200 10 0.2 6.0 67
    36 212 10 1.2 9.7 88
    42 202 2 0.1 15.5 98
    48 197 12 1.2 23.8 112
  • Similar results were obtained with an identical vitamin B[0307] 12 feed at half the concentration or bolus additions of vitamin B12 across the time course of the fermentation. The highest titer obtained was 114 g/L.
    • EXAMPLE 2A Comparison of 1,3-propanediol and Glycerol Production with E. coli Strains RJ8/pAH48/pDT29 and RJ8/pAH48/DKP32
  • RJ8/pAH48pDT29 and RJ8/pAH48/pKP32 pre-cultures were prepared using frozen vials thawed and grown in 500 mL 2YT with 200 mg/L carbenicillin and 50 mg/L spectinomycin. The full contents of the flask were used to inoculate the fermenter. The fermenter was operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods. RJ8/pAH48/pKP32 is identical to RJ8/pAH48/pDT29 except that dhaT is deleted. The vitamin B[0308] 12 strategy for each fermentation is detailed below.
  • Fermentation with RJ8/pAH48/pDT29. [0309]
  • A representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using [0310] E. coli strain RJ8/pAH48/pDT29 is given in Table 2A.1. Vitamin B12 was provided as bolus additions of 2, 16 and 16 mg at 2, 8, and 26 h, respectively. The yield of 1,3-propanediol was 35 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 50.1 g/L 1,3-propanediol was obtained.
    TABLE 2A.1
    Representative fermentation summary of the
    conversion of glucose to 1,3-propanediol (1,3-PD)
    using E. coli strain RJ8/pAH48/pDT29
    Time OD550 DO Glucose Glycerol 1,3-PD
    (h) (AU) (%) (g/L) (g/L) (g/L)
    0 0 140 10.6 0.1 0.0
    6 5 107 11.1 0.5 0.4
    10 16 90 8.5 1.7 1.3
    14 25 86 1.8 2.4 5.9
    19 38 53 3.5 5.9 15.4
    25 53 38 0.1 9.2 26.7
    31 54 10 4.5 7.4 39.0
    37 37 23 17.2 6.0 45.0
    43 21 13 9.9 7.7 50.1
  • Improved Fermentation with RJ8/pDAH48/pKP32. [0311]
  • A representative fermentation summary of the conversion of glucose to 1,3-propanediol (1,3-PD) using [0312] E. coli strain RJ8/pAH48/pKP32 is given in Table 2A.2. Vitamin B12 was provided as bolus additions of 48 and 16 mg at approximately 26 and 44 hr, respectively. The yield of 1,3-propanediol was 34 wt % (g 1,3-propanediol/g glucose consumed) and a titer of 129 g/L 1,3-propanediol was obtained.
    TABLE 2A.2
    Representative fermentation summary of the improved
    conversion of glucose to 1,3-propanediol (1,3-PD)
    using E. coli strain RJ8/pAH48/pKP32.
    Time OD550 DO Glucose Glycerol 1,3-PD
    (h) (AU) (%) (g/L) (g/L) (g/L)
    0 0 150 12.6 0.1 0
    6 12 113 6.0 2.6 0
    12 24 99 0.0 10.6 0
    18 51 76 2.4 28.9 0
    24 78 82 2.4 44.2 5
    30 114 70 3.8 26.9 33
    36 111 72 0.0 20.0 57
    42 139 65 0.1 21.9 69
    48 157 36 0.1 22.4 79
    55 158 25 0.2 21.4 94
    64 169 14 0.1 15.8 113
    72 169 12 0.1 13.4 119
    74 162 14 0.1 14.8 129
    • 1,3-Propanediol and Glycerol Production with E. coli Strain RJ8. n/pAH48/pKP32.
  • Strain RJ8.n was transformed with plasmids pAH48 and pKP32. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods. A thawed frozen vial of RJ8.n/pAH48/pKP32 was transferred to 500 mL LB with 200 mg/L carbenicillin and 50 mg/L spectinomycin to prepare the pre-culture. The culture was transferred to a seed fermenter and grown for 16 h before 1 L of the culture was transferred to the production fermenter. At that time, the OD550 had reached over 50 AU and 30 g/L glycerol had accumulated in the broth. Both the seed and production fermenters were operated at 35° C. and a d6 set-point of 10%; all other control parameters are as described in General Methods. [0313]
  • Vitamin B[0314] 12 was added to the production tank in 16 mg boluses at 12 h, 17.3 h, 22.8 h, and 27.5 h. The final titer was 112.7 g/L 3G and the mass yield was 31.6%.
    • EXAMPLE 3 Construction and Shake Flask Performance of an E. coli Strain with a Deletion in the arcA Global Regulator
  • An arcA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 0.6 kb of the coding region with the FRT-CmR-FRT cassette of pKD3. A replacement cassette was amplified with the primer pair SEQ ID NO:2 and SEQ ID NO:3 using pKD3 as the template. The primer SEQ ID NO:2 contains 41 bp of homology to the 5′ end of arcA and 20 bp of homology to pKD3. Primer SEQ ID NO:3 contains 42 bp of homology to the 3′ end of arcA and 20 bp of homology to pKD3. The PCR product was gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of chloramphenicol. The deletion of the arcA gene was confirmed by PCR, using the primer pair SEQ ID NO:4 and SEQ ID NO:5. The wild-type strain gives a 0.7 kb PCR product while the recombinant strain gives a characteristic 1.1 kb PCR product. The strain has been designated MG1655 AarcA::Cm. A P1 lysate was prepared and used to move the mutation into the strain KLndh81 to form KLndh81 ΔarcA::Cm. [0315]
  • The KLndh81 ΔarcA::Cm strain and KLndh81 control strain were electrotransformed with plasmid pSYCO101. One colony of each strain was incubated 10 h in LB medium with 50 mg/L spectinomycin. A 200 μL volume of these cultures was then transferred to a 250 mL baffled Erlenmeyer flask containing 10 mL TM2 medium with 40 g/L glucose, 50 mg/L spectinomycin, and 2 mg/L vitamin B[0316] 12. The flasks were incubated at 300 rpm and 34° C. for 40 hrs. The results in Table 3 show that the arcA mutation improves the molar yield of glycerol and 1, 3 propanediol production.
    TABLE 3
    Glycerol and 1,3-propanediol production in E. coli
    control and ΔarcA::Cm strains
    1,3
    Glycerol Propanediol OD Molar Yield
    Strain (g/L) (g/L) 550 nm (mol/mol)
    KLndh81 pSYCO101 5.8 10.7 29.2 0.87
    KLndh81 ΔarcA 6.8 11.4 25.9 0.95
    pSYCO101
    • EXAMPLE 4 Construction of Phosphotranserase System Minus (PTS−) E. coli Strains with TRC Promoters Controlling Expression Levels of Galactose-Proton Symporter (galP) and Glucokinase (qlk)
  • Construction of a loxP-CAT-loxP-Trc Cassette (pTrCm42). [0317]
  • Linear DNA was obtained from plasmid pTrc99a (Pharmacia) digested with HindIII and NcoI, filled with T4 DNA polymerase, circularized and transformed into [0318] E. coli TOP-10 (Invitrogen, Carlsbad, Calif.). Following selection on Luria-agar plates containing 50 mg/L of carbenicillin, the resulting plasmid (pTrcl) was purified and subjected to restriction enzyme analysis to confirm that the DNA region originally present between HindIII and NcoI was absent.
  • The unique BspM1 site in pTrc1 (upstream of the −35 region of the trc promoter) was used to insert a cassette containing a chloramphenicol resistance gene (CAT) flanked by loxP sites. Linear DNA was obtained from pTrc1 digested with BspM1, gel-purified using a QIAquick gel extraction kit (QIAGEN), filled in with T4 DNA polymerase, and ligated to a loxP-Cat-loxP cassette. The loxP-Cat-loxP cassette was obtained from the plasmid pLoxCat1, see SEQ ID NO:6, by digestion with Stu1 and BamH1. pLoxCat1 is similar to pLoxCat2 (Palmeros et al, supra]. The ligation mixture was transformed into [0319] E. coli TOP-10 (Invitrogen) and selection was performed on Luria-agar plates containing 50 mg/L of carbenicillin and 20 mg/L of chloramphenicol. Plasmid was obtained and restriction enzyme analysis performed. Two plasmids, containing loxP-Cat-loxP-Trc with the loxP-Cat-loxP cassette in the same and in the opposite orientation relative to the trc promoter, were designated pTrCm41 and pTrCm42, respectively.
  • Construction of a trc Promoter Replacement Template for galP (pR6KqalP). [0320]
  • A DNA cassette containing the trc promoter and lac operator with an upstream loxP-CAT-loxP cassette was PCR amplified from pTrCm42 using the primer pair SEQ ID NO:7 and SEQ ID NO:8. The primer pair incorporates 40 bp of homology to the galP upstream region to each end of the PCR product. PCR parameters were 95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min, 30 cycles using Taq polymerase (Roche). The product was subcloned into Echo pUni/His5 R6K (Invitrogen) to generate the plasmid pR6 KgalP. [0321]
  • Construction of a trc Promoter Replacement Template for qlk (pR6Kglk). [0322]
  • A DNA cassette containing the trc promoter and lac operator with an upstream loxP-CAT-loxP cassette was amplified from pTrCm42 by PCR using the primer pair SEQ ID NO:9 and SEQ ID NO:10. The primer pair SEQ ID NO:9 and SEQ ID NO:10 incorporates 39 (with a one base deletion) and 40 bp of homology, respectively, to the glk upstream region to each end of the PCR product. PCR parameters were 95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min, 30 cycles using Taq polymerase (Roche). The product was subcloned into Echo pUni/His5 R6K (Invitrogen) to generate the plasmid pR6 Kglk. [0323]
  • Construction of an [0324] E. coli AptsHicrr Strain (KLpts7).
  • A PTS minus derivative (AptsHIcrr) of [0325] E. coli strain KLndh81 was obtained by P1 vir transduction using a derivative of E. coli strain NF9 as donor (Flores et al., Nature Biotechnology 14, 620-623 (1996)). The transduction replaces the operon comprising ptsH, ptsl and crr with a kanamycin antibiotic resistance marker (Levy et al., Gene 86, 27-33 (1990)) to give strain KLpts7. Plated on MacConkey (lactose−) agar +1% glucose, KLpts7 exhibits a white colony phenotype.
  • Replacement of the Natural galP Promoter with the Synthetic trc Promoter (KLqalP-trc). [0326]
  • A PCR amplification product comprising a loxP-Cat-loxP-Trc cassette and incorporating 40 bp of homology to the galP upstream region [for reference, see GenBank, Accession # U00096] to each end was generated using rtth RNA polymerase (Perkin Elmer), pR6 KgalP as the template and the primer pair SEQ ID NO:7 and SEQ ID NO:8. The PCR amplified integration cassette was transformed into electro-competent KLpts7 cells containing pKD46 for integration using the lambda Red system as described in Datsenko and Wanner, supra. Selection was performed on LB plates containing 10 mg/L chloramphenicol. Successful integration of this cassette replaces the region 38 to 181 bp upstream of the galP ATG start codon (for reference, see GenBank, Accession # U00096) with a loxP-Cat-loxP-Trc cassette (SEQ ID NO:11) to provide strain KLpts::galP-trc. Integration was confirmed by PCR analysis using the primer pair SEQ ID NO:7 and SEQ ID NO:8 (amplifying the integration site to give a 1.4 kb product) and the primer pair SEQ ID NO:12 and SEQ ID NO:13 (amplifying the integration site, including upstream and downstream regions, to give a 2.1 kb product). PCR parameters were 95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min, 30 cycles using Taq polymerase or rtth polymerase. KLpts::galP-trc, plated on MacConkey (lactose[0327] −) agar +1% glucose, exhibits a light red colony phenotype. The chloramphenicol marker was removed as described by Palmeros et al., supra. The removal was confirmed by PCR analysis using the primer pair SEQ ID NO:12 and SEQ ID NO:13 (to give a 1.1 kb product) and the resulting strain was designated KLgaIP-trc.
    • Replacement of the Natural glk Promoter with the Synthetic trc Promoter (KLGG)
  • A PCR amplification product comprising a loxP-Cat-loxP-Trc cassette and incorporating approximately 40 bp of homology to the glk upstream region [for reference, see GenBank, Accession # U00096] to each end was generated using rtth RNA polymerase (Perkin Elmer), pR6 Kglk as the template, and the primer pair SEQ ID NO:9 and SEQ ID NO:10. The PCR amplified integration cassette was transformed into electro-competent KLgaIP-trc cells containing pKD46 for integration using the lambda Red system as described above. Selection was performed on LB plates containing 10 mg/L chloramphenicol. Successful integration of this cassette replaces the [0328] region 40 to 137 bp upstream of the glk ATG start codon (for reference, see GenBank, Accession # U00096) with a loxP-Cat-loxP-Trc cassette (SEQ ID NO:11). Integration was confirmed by PCR analysis using the primer pair SEQ ID NO:14 and SEQ ID NO:15 (amplifying the integration site, including upstream and downstream regions, to give a 2.4 kb product). Plated onto MacConkey (lactose−) agar +1% glucose, colonies exhibit a deep red color, indicating an increase in the conversion of glucose to acid compared to the parent (KLgalP-trc). The chloramphenicol marker was removed as described above and subsequent PCR analysis (using the primer pair SEQ ID NO:14 and SEQ ID NO:15 to give a 1.3 kb product) gave the strain KLGG.
  • Deletion of the arcA Gene, Encoding a Global Regulator (KLGG AarcA). [0329]
  • A P1 lysate of the MG1655 ΔarcA::Cm strain was prepared and used to move the mutation to strain KLGG. A resulting chloramphenicol resistant clone, KLGG ΔarcA::Cm, was checked by genomic PCR to insure that the mutation was present. The chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) and this strain has been designated KLGG ΔarcA. [0330]
  • Deletion of the edd Gene, Encoding 6-phosphogluconate Dehydrase Gene (KLGG ΔarcA Δedd, also Designated FMP). [0331]
  • An edd deletion [for reference, see GenBank, Accession # U00096] was obtained by replacing 1.7 kb of the coding region with a loxP-cat-loxP cassette from pLoxCat2. A replacement cassette was amplified with the primer pair SEQ ID NO:16 and SEQ ID NO:17. The primer SEQ ID NO:16 contains 80 bp of homology to the 5′ end of edd and 18 bp of homology to the template pLoxCat2. The primer SEQ ID NO:17 contains 78 bp of homology to the 3′ end of edd and 19 bp of homology to pLoxCat2. The PCR product was gel-purified and electroporated into KLGG AarcA/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol. The deletion of the edd gene was confirmed by PCR using primer pair SEQ ID NO:18 and SEQ ID NO:19. The wild-type strain gives a 2.2 kb PCR product while the recombinant gives a characteristic 1.6 kb PCR product. This strain has been designated KLGG ΔarcA Δedd::cat. The chloramphenicol marker was removed using the Cre recombinase (Palmeros et al., supra) and this strain has been designated KLGG ΔarcA Δedd or, alternatively, FMP. [0332]
    • Selection and Characterization of an FMP Strain Exhibiting an Enhanced Rate of Glucose Consumption
  • Invariably, cells comprising ΔptsHlcrr, the trc promoter replacement of the natural galP promoter, and the trc promoter replacement of the natural glk promoter (all three modifications as described in Example 4) initially exhibited slow growth. Also invariably, a subsequent selection (as described below) lead to a faster growing derivative. Glucokinase activity, assayed from cell-free extracts, was typically three-fold higher for the faster growing derivative as compared to the slower growing parent. [0333]
  • [0334] E. coli strain FMP, transformed with plasmid pSYCO103, was grown in a 14 L fermenter essentially as described in Example 2. Vials for storage at −80° C. (15% glycerol stocks) were made over the course of the fermentation. A LB plate was streaked from the aliquot taken at 33 hrs (OD550 was 30.7 AU) and single colonies were recovered and designated “selected FMP/pSYCO103”. Single colonies were similarly obtained from strain FMP containing no plasmid and designated “selected FMP”.
  • The galP and glk genes, including the introduced trc promoter region, were PCR amplified from two colonies of “selected FMP/pSYCO103” and one colony of “selected FMP” using the primer pair SEQ ID NO:12/SEQ ID NO:13 and the primer pair SEQ ID NO:14/SEQ ID NO:15, respectively. Sequence analysis on these three samples was performed using the same primers. In each case, the galP gene and promoter region remained unchanged from the parent strain while the glk gene and promoter region contained an identical, single base pair change when compared the parent strain. The two isolates of “selected FMP/pSYCO103” and one isolate of “selected FMP” contained the sequence identified as SEQ ID NO:20 in the −35 to −10 region of trc promoter controlling glk expression compared to the corresponding parent sequence SEQ ID NO:21. The strain originating from “selected FMP” from which galP and glk sequence was obtained was designated strain FMP′::Km. [0335]
    • EXAMPLE 5 Comparative Examples of Glucose to 1,3-propanediol Fermentations Using Strains FMP/pSYCO103 and FMP′::Km/pSYCO103
  • Strains FMP and FMP′::Km were transformed with the plasmid pSYCO103. Production of 1,3-propanediol was determined in 14 L fermenters as described in General Methods with the following differences in the control parameters or the fermenter. A thawed frozen vial of FMP/pSYCO103 was transferred to 500 mL 2YT with 50 mg/L spectinomycin to prepare the pre-culture. The dO set-point was 15%. Vitamin B[0336] 12 was added to the fermenter in 16 mg boluses at 30, 43, and 51 h.
  • A thawed frozen vial of FMP′::Km/pSYCO103 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture. Vitamin B[0337] 12 was added to the fermenter in 16 mg boluses at 21, 40 and 40.5 h.
  • FIG. 1 shows the production of 1,3-propanediol by FMP′::Km/pSYCO103 is faster than by FMP/pSYCO103. [0338]
    • EXAMPLE 6 Engineering Altered Expression of Glyceraldehyde-3-phosphate Dehydrogenase (gapA) in E. coli Strains for Production of 1,3 Propanediol from Glucose
  • Decreasing GaPA expression by altering the start condon. [0339]
  • The level of glyceraldehyde-3-phosphate dehydrogenase, GapA, was decreased by replacing the ATG start codon of the gapA gene with a GTG or TTG start codon. [0340]
  • The [0341] E. coli gapA gene plus upstream and downstream flanking sequence was amplified by PCR from E. coli strain MG1655 using primer pair SEQ ID NO:22 and SEQ ID NO:23. The PCR product was cloned directly from the PCR reaction into pCR-BluntII-TOPO (Invitrogen) to form pDT50. The plasmids pDT50 and pLitmus 28 (New England Biolabs, Inc.) were digested with SphI and BamH I and the gapA gene fragment and vector, respectively, were gel-purified and ligated. The resulting plasmid, pDT51, was transformed into E. coli TOP10 (Invitrogen).
  • The gapA mutant plasmids were constructed using the Stratagene QuickChange 1-Day Site-Directed Mutagenesis Method (Stratagene, La Jolla, Calif.). The template plasmid, pDT51, was mixed with either the primer pair SEQ ID NO:24 and SEQ ID NO:25 to create the GTG mutation or the primer pair SEQ ID NO:26 and SEQ ID NO:27 to create the TTG mutation. After the PCR amplification, the reactions were digested with Dpnl to remove the template plasmid and leave only the amplified plasmids. The plasmids were then transformed into [0342] E. coli TOP10 (Invitrogen).
  • The gapA-GTG and gapA-TTG constructs were PCR amplified using primer pair SEQ ID NO:22 and SEQ ID NO:23. The PCR products were electroporated into the gapA knockout strain, KLP23A112. [0343] E. coli strain KLP23A112 was constructed by transducing KLP23 with a P1 phage lysate obtained from E. coli DS112 (E. coli Genetic Stock Center), a gapA deletion strain containing a CmR marker. Recombinants were selected for growth on LB plates without added glucose and sensitivity to chloramphenicol. Sequencing confirmed the successful integration of the GTG and TTG mutations. The mutated strains were named KLPAGTG and KLPATTG, respectively. Measurement of the GapA activities in the KLPAGTG and KLPATTG strains showed that the GapA levels were 4% and <1% of the control strain KLP23, respectively.
  • Strains KLP23, KLPAGTG, and KLPATTG were transformed with the plasmid pSYCO101 and tested for 1,3-propanediol production in TM2 medium containing 40 g/L glucose, 50 mg/L spectinomycin, and 1 mg/L vitamin B[0344] 12. The results are shown in Table 4.
    TABLE 4
    Representative shake flasks summary of the conversion of
    glucose to 1,3-propanediol using E. coli strains KLP
    23/pSYCO101, KLP AGTG/pSYCO101, and
    KLPATTG/pSYCO101
    Glycerol 1,3-Propanediol Molar Yield
    Strain (g/L) (g/L) (mol/mol)
    KLP23 pSYCO101 5.8 10.7 0.87
    KLPAGTG pSYCO101 0.7 1.3 0.11
    KLPATTG pSYCO101 0.2 0.4 0.04
    • Altering GapA Expression by Replacing the Promoter
  • Replacement of the natural gapA promoter with the synthetic short 1.5 GI promoter (SEQ ID NO:28) was made by replacing 225 bp of upstream gapA sequence [for reference, see GenBank, Accession # U00096] with FRT-CmR-FRT and an engineered promoter. The replacement cassette was amplified by PCR with the primer pair SEQ ID NO:29 and SEQ ID NO:30 using pKD3 as a template. The primer SEQ ID NO:29 contains 39 bp of homology to gapA including the ATG start, contains the short 1.5 GI promoter and contains 20 bp of homology to template pKD3. Primer SEQ ID NO:30 contains 59 bp of homology to upstream gapA sequence and 21 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells to give MG1655 1.5gapA::Cm. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol. Successful integration of the cassette replaces the region 34-258 bp upstream of the gapA ATG start codon with a FRT-CmR-FRT-short 1.5 GI promoter cassette. A P1 phage lysate was prepared and used to move the mutation to FMP′::Km. This strain was designated FMP′::Km 1.5gapA::Cm. [0345]
  • The short 1.5 GI gapA promoter in MG1655 1.5gapA::Cm was replaced with the short 1.20 GI promoter (SEQ ID NO:31) or the short 1.6 GI promoter (SEQ ID NO:32). To create the 1.20 gapA strain, a replacement cassette was PCR amplified with primer pair SEQ ID NO:33 and SEQ ID NO:34 using genomic DNA from MG1655 1.5gapA::Cm as template. Primer SEQ ID NO:33 contains 24 bp of homology to the gapA upstream region. Primer SEQ ID NO:34 contains homology to the gapA upstream region in MG1655 1.5gapA::Cm and contains the short 1.20 GI promoter. To create the 1.6gapA strain, a replacement cassette was PCR amplified with primer pair SEQ ID NO:33 and SEQ ID NO:35 using genomic DNA from MG1655 1.5gapA::Cm as template. The primer SEQ ID NO:35 contains homology to the gapA upstream region in MG1655 1.5gapA::Cm and contains the short 1.6 GI promoter. The short 1.20 GI promoter replacement cassette and the short 1.6 GI promoter replacement cassette were used to replace the natural gapA promoter of MG1655 as described above to give strains MG1655 1.20gapA::Cm and MG1655 1.6gapA::Cm, respectively. MG1655 1.20gapA::Cm and MG1 655 1.6gapA::Cm were used to replace the natural gapA promoter of strain FMP′::Km (using P1 tranduction as described above) to give strains FMP′::Km 1.20gapA::Cm and FMP′::Km 1.6gapA::Cm, respectively. [0346]
  • Glyceraldehyde-3-phosphate dehydrogenase activities were determined using cell-free extracts prepared from the strains FMP′::Km 1.20gapA::Cm, FMP′::Km 1.5gapA::Cm, FMP′::Km 1.6gapA::Cm and FMP′::Km as control. The values obtained, compared to that of control, were 10%, 25% and 140% for strains FMP′::Km 1.20gapA::Cm, FMP′::Km 1.5gapA::Cm, FMP′::Km 1.6gapA::Cm, respectively. [0347]
  • The strains containing the GI promoter replacements were transformed with the plasmid pSYCO106 and compared to the parent strain for 1,3-propanediol production in TM2 medium containing 20 g/L glucose, 50 mg/L spectinomycin, and 1 mg/L vitamin B[0348] 12. The results are shown in Table 5.
    TABLE 5
    Representative shake flasks summary of FMP'::Km GI promoter
    gapA strains transformed with the plasmid pSYCO106
    1,3- Molar
    Glycerol Propanediol yield
    Strain (g/L) (g/L) (mol/mol)
    FMP'::Km/pSYCO106 1.8 6.8 1.07
    FMP'::Km 1.6 gapA/pSYCO106 4.1 3.3 0.92
    FMP'::Km 1.5 gapA/pSYCO106 6.6 3.6 1.21
    FMP'::Km 1.20 gapA/pSYCO106 7.6 2.8 1.26
    • EXAMPLE 7 Removing the Markers from FMP′::Km 1.5gapA::Cm
  • The chloramphenicol marker was removed from strain FMP′::Km 1.5gapA::Cm (as described in the General Methods section) to give strains FMP′::Km 1.5gapA. [0349]
  • The kanamycin marker introduced into FMP′::Km 1.5gapA as a consequence of making KLndh81 PTS minus (ΔptsHlcrr) was replaced with a removable FRT-Cm-FRT cassette by P1 transduction from MG1655 ΔptsHlcrr::Cm. A ptsHlcrr deletion in MG1655 was made with a replacement cassette amplified with primer pair SEQ ID NO:54 and SEQ ID NO:55 using pKD3 as template. The primer SEQ ID NO:54 contains 78 bp of homology to the remaining region of ptsH left in the chromosome of strain FMP′::Km 1.5gapA and 20 bp of homology to pKD3. The primer SEQ ID NO:55 contains 77 bp of homology to the remaining region of crr in strain FMP′::Km 1.5gapA and 20 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with chloramphenicol 12.5 mg/L. PCR analysis confirmed the integration of the cassette. Plated on MacConkey (lactose[0350] −) agar +1% glucose, MG1655 ΔptsHlcrr::Cm exhibits a white colony phenotype. A P1 phage lysate was prepared and the Cm marker transduced into FMP′::Km 1.5gapA. Recombinant strains were selected on chloramphenicol and PCR analysis confirmed successful integration of the cassette. The chloramphenicol marker was removed using the Flp recombinase and sequencing (using primer SEQ ID NO:56) confirmed the removal of the chloramphenicol marker. The resulting strain was designated FMP′1.5 gapA.
    • EXAMPLE 8 Demonstrating High Yield of 1,3-propanediol from Glucose using E. coli Strain FMP′ 1.5gaDA/pSYCO106
  • Strain FMP′ 1.5gapA was transformed with the plasmid pSYCO106. Production of 1,3-propanediol and glycerol was determined in 14 L fermenters as described in General Methods with the following differences in the control parameters for the fermenter. A thawed frozen vial of FMP′ 1.5gap/pSYCO106 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture. Vitamin B[0351] 12 was added to the fermenter in 16 mg boluses prior to inoculation and at 28 hrs. Final 1,3-propanediol concentration was 129 g/L and the mass yield was 40.2%.
    • EXAMPLE 9 Engineering a Methylglyoxal Synthase (mgsA) Mutant in E. coli
  • The mgsA deletions [for reference, see GenBank, Accession # U00096] were made by replacing 0.4 kb of the coding region with the FRT-Kan-FRT cassette of pKD4. A replacement cassette was PCR amplified with the primer pair SEQ ID NO:36 and SEQ ID NO:37 using pKD4 as the template. The primer SEQ ID NO:36 contains 40 bp of homology to the 5′ end of mgsA and 20 bp of homology to the template DNA, pKD4. The primer SEQ ID NO:37 contains 40 bp of homology to the 3′ end of mgsA and 20 bp of homology to pKD4. The PCR product was gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of kanamycin. The deletion of the mgsA gene was confirmed by PCR, using the primer pair SEQ ID NO:38 and SEQ ID NO:39. The wild-type strain gives a 1.3 kb PCR product while the recombinant strain gives a characteristic 2.4 kb PCR product. This strain has been designated MG1655 ΔmgsA::kan. Once the mgsA mutant was obtained in MG1655, a P1 phage lysate was prepared and used to move the mutation into FMP′ 1.5gapA (Example 8). The kanamycin resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) and this strain has been designated FMP′ 1.5gapA AmgsA. [0352]
  • FMP′ 1.5gapA ΔmgsA and its parent were transformed with the plasmid pSYCO106 to give FMP′1.5gapA ΔmgsA/pSYCO106 and FMP′ 1.5gapA/pSYCO106, respectively. [0353]
  • The strains were tested for 1,3-propanediol production in TM2 medium containing 20 g/L glucose, 50 mg/L spectinomycin, and 1 mg/L vitamin B[0354] 12. The results are shown in Table 6.
    TABLE 6
    Representative shake flasks summary of FMP' 1.5
    gapA/pSYCO106 and FMP' 1.5 gapA
    ΔmgsA/pSYCO106 strains
    1,3- Molar
    Glycerol Propanediol yield
    Strain (g/L) (g/L) (mol/mol)
    FMP'1.5 gapA/pSYCO106 6.6 3.6 1.21
    FMP'1.5 gapA ΔmgsA/pSYCO106 8.3 2.4 1.26
    • EXAMPLE 10 Fermentation of Glucose to 1,3-propanediol using E. coli Strain FMP′ 1.5gapA ΔmgsA/pSYCO106
  • Strain FMP′ 1.5gapA ΔmgsA was transformed with the plasmid pSYCO106. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermenters. A single colony from a fresh plate (LA with 50 mg/L spectinomycin) of FMP′ 1.5gapA ΔmgsA pSYCO106 was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to prepare the pre-culture. After incubation at 34° C. and 300 rpm to an OD[0355] 550 of 1 AU, 10.8 mL of the culture was transferred to the fermenter. The fermenter was run with glucose limitation during much of the run. Vitamin B12 was added to the fermenter in 16 mg boluses prior to inoculation, at 28 hrs and at 38 hrs. Final 1,3-propanediol concentration was 130 g/L and the mass yield was 47.5%. A run with glucose maintained in excess (0-20 g/L) gave 141 g/L 1,3-propanediol and a mass yield of 43.6%.
    • EXAMPLE 11 Construction of an E. coli Strain with an Engineered Promoter for Phosphoenolpyruvate Carboxylase (PpC) by Linear DNA Transformation
  • Replacement of the natural ppc promoter with the synthetic short 1.6 GI promoter was made by replacing 59 bp of the upstream ppc sequence cassette containing FRT-CmR-FRT and an engineered promoter. The PCR product was amplified with the primer pair SEQ ID NO:40 and SEQ ID NO:41 using pKD3 as the template. Primer SEQ ID NO:40 contains 80 bp of homology to upstream ppc sequence and 20 bp of homology to template pKD3. Primer SEQ ID NO:41 contains 39 bp of homology to upstream ppc sequence, contains the short 1.6 GI promoter sequence and contains 20 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol to give MG1655 1.6 ppc::Cm. Successful integration of the cassette replaces the region 90 to 148 bp upstream of the ppc ATG start [for reference, see GenBank, Accession # U00096] with a FRT-CmR-FRT-short 1.6 GI promoter cassette. Integration into the upstream ppc region was confirmed by primer pair SEQ ID NO:40 and SEQ ID NO:41. The wild-type strain gives a 0.2 kb PCR product while the recombinant strain gives a characteristic 1.2 kb PCR product. This PCR product was sequenced using primer SEQ ID NO:42, which indicated that the promoter replacement effectively occurred. A P1 phage lysate was prepared and used to move the mutation to strain FMP′1.5gap ΔmgsA. This strain was designated FMP′1.5gap Δmgs 1.6 ppc::Cm. The chloramphenicol-resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra), and the resulting strain was electrotransformed with plasmid pSYCO106 to give FMP′1.5gap Δmgs 1.6 ppc/pSYCO106. [0356]
  • Shake flask cultures were used to assess the conversion of glucose to 1,3-propanediol in [0357] E. coli strains FMP′1.5gap Δmgs/pSYCO106 and FMP′1.5gap Δmgs 1.6 ppc/pSYCO106. The strains, grown in LB medium containing 50 mg/L spectinomycin for 10 hrs, were used to inoculated (200 μL) into 250 mL-baffled Erlenmeyer flasks containing 10 mL TM2 medium, 20 g/L glucose, 50 mg/L spectinomycin, and 2 mg/L vitamin B12. The flasks were incubated at 300 rpm and 34° C. Representative results are given in Table 7. Both an increase in the molar yield and a decrease of acetate production were observed with the addition of the 1.6 ppc mutation to the parent strain.
    TABLE 7
    Shake Flasks for Conversion of Glucose to
    1,3-Propanediol (1,3-PD)
    Glycerol 1,3- Acetate
    produced Propanediol produced Molar
    Strain (g/L) produced (g/L) (g/L) Yield
    FMP'1.5 gap 8.24 2.19 1.78 1.25
    ΔmgsA/pSYCO106
    FMP'1.5 gap Δmgs 7.5 3.34 0.34 1.32
    1.6 ppc/pSYCO106
  • Phosphoenolpyruvate carboxylase (Ppc) activity was measured from cell-free extracts obtained from the shake flasks described immediately above. Aliquots of cells were harvested in mid-log phase, broken by two passages through a French press cell, centrifuged for 15 15 min at 14,000 rpm, and ultracentrifuged 1 hr at 50,000 rpm. The supernatant was removed and used as a source of proteins. Specific activities of PPC are reported in Table 8 below. The replacement of the natural ppc promoter with the short 1.6 GI promoter increased the Ppc enzyme activity three-fold. [0358]
    TABLE 8
    PPC Enzyme Specific Activity
    Phosphoenolpyruvate
    carboxylase
    Strain specific activity (U/mg protein)
    FMP′ 1.5 gap ΔmgsA, pSYCO106 0.28
    FMP′ 1.5 gap Δmgs 1.6 ppc, 0.86
    pSYCO106
    • EXAMPLE 11A Fermentation of Glucose to 1,3-propanediol using E. coli Strain FMP′ 1.5gapA ΔmgsA/PSYCO106
  • Production of 1,3-propanediol by FMP′ 1.5gapA ΔmgsA 1.6 ppc/pSYCO106 was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermenters. A single colony from a fresh plate (LA with 50 mg/L spectinomycin) of FMP′ 1.5gapA ΔmgsA 1.6 ppc/pSYCO106 was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to prepare the pre-culture. After incubation at 34° C. and 300 rpm to an OD550 of 1 AU, 10.8 mL of the culture was transferred to the fermenter. Vitamin B[0359] 12 was added to the fermenter in 16 mg boluses prior to inoculation, at 28 hrs, and at 38 hrs. Final 1,3-propanediol concentration was 135.3 g/L and the mass yield was 46.1%.
    • EXAMPLE 12 Construction of E. coli Strain with an Engineered Promoter for yciK/btuR by Linear DNA Transformation
  • The genes yciK and btuR are present within a single operon in [0360] E. coli. Replacement of the natural yciK-btuR promoter with the synthetic short 1.6 GI promoter was made by inserting a 1.3 kb cassette, upstream of yciK-btuR. The replacement cassette, containing FRT-CmR-FRT and an engineered promoter, was amplified by PCR with the primer pair SEQ ID NO:43 and SEQ ID NO:44 using pKD13 as the template. Primer SEQ ID NO:43 contains 70 bp of homology to upstream yciK-btuR sequence and 20 bp of homology to template pKD13. Primer SEQ ID NO:44 contains 30 bp of homology to upstream yciK-btuR sequence, contains the short 1.6 GI promoter sequence and contains 20 bp of homology to pKD13. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 25.0 mg/L kanamycin to give MG1655 1.6yciK-btuR::Km. Successful integration of the cassette results in an insertion between bp 27 and bp 28 upstream of the ycik ATG start codon [for reference, see GenBank, Accession # U00096] with a FRT-CmR-FRT-short 1.6 GI promoter cassette. Integration into the upstream yciK/btuR region was confirmed by primer pair SEQ ID NO:45 and SEQ ID NO:46. The wild-type strain gives a 1.4 kb PCR product while the recombinant strain gives a characteristic 2.8 kb PCR product. A P1 phage lysate was prepared and used to move the mutation to a derivative of strain FMP′1.5gap ∴mgs 1.6 ppc named Triple. After removing the antibiotic, strain Triple 1.6btuR was obtained.
    • EXAMPLE 12A Fermentation of Glucose to 1,3-propanediol using E. coli Strain Triple 1.6btuR/pSYCO109
  • Strain Triple 1.6btuR was transformed with pSYCO109. Production of 1,3-propanediol by Triple 1.6btuR/pSYCO109 was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermentation. A single colony from a fresh plate (LA with 50 mg/L spectinomycin) of Triple 1.6btuR/pSYCO109 was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to prepare the pre-culture. After incubation at 34° C. and 300 rpm to an OD550 of 1 AU, 10.8 mL of the culture was transferred to the fermenter. Vitamin B[0361] 12 was added to the fermenter in 8 mg boluses prior to inoculation, at 28 hrs, and at 38 hrs. Final 1,3-propanediol concentration was 123 g/L and the mass yield was 45.7%.
    • EXAMPLE 13 Construction of E. coli Strain with an Engineered Promoter for vqhD by Linear DNA Transformation
  • Replacement of the natural yqhD (alcohol dehydrogenase) promoter with the synthetic short 1.6 GI promoter was made by replacing 967 bp of upstream yqhD sequence including the yqhc gene with a cassette containing FRT-CmR-FRT and an engineered promoter. The PCR product was amplified with the primer pair SEQ ID NO:47 and SEQ ID NO:48 using pKD3 as the template. Primer SEQ ID NO:47 contains 78 bp of homology to upstream yqhD sequence and 20 bp of homology to template pKD3. Primer SEQ ID NO:48 incorporates 41 bp of homology (with a 1 bp deletion) to upstream yqhD sequence, 40 bp of homology to the short 1.6 GI promoter sequence, and 19 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L chloramphenicol to give MG1655 1.6yqhD::Cm. Successful integration of the cassette replaces the region 50-1016 bp upstream of the yqhD ATG start [for reference, see GenBank, Accession # U00096] with a FRT-CmR-FRT-short 1.6 GI promoter cassette. Integration into the upstream yqhD region was confirmed by sequencing with primer SEQ ID NO:49 and indicated that the promoter replacement effectively occurred. A P1 phage lysate was prepared and used to move the mutation into strain Triple 1.6btuR. After removing the antibiotic (as described above), strain Triple 1.6btuR 1.6yqhD was obtained. [0362]
    • EXAMPLE 13A Fermentation of Glucose to 1,3-propanediol using E. coli Strain Triple 1.6btuR 1.6vqhD/pSYCO109
  • Strain Triple 1.6btuR 1.6yqhD was transformed with pSYCO109. Production of 1,3-propanediol by Triple 1.6btuR 1.6yqhD/pSYCO109 was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermentation. A single colony from a fresh plate (LA with 50 mg/L spectinomycin) of Triple 1.6btuR 1.6yqhD/pSYCO109 was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to prepare the pre-culture. After incubation at 34° C. and 300 rpm to an OD550 of 1 AU, 10.8 mL of the culture was transferred to the fermenter. Vitamin B[0363] 12 was added to the fermenter in a single 16 mg bolus at 20.6 hrs elapsed fermentation time. Final 1,3-propanediol concentration was 113.3 g/L and the mass yield was 48.8%.
    • EXAMPLE 14 Construction of an E. coli Strain with a Deletion Mutation in Acetate Kinase (ack) and Phosphotransacetylase (pta) by Linear DNA Transformation
  • The pta-ackA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 3.3 kb of the coding region with the FRT-CmR-FRT cassette of pKD3. The replacement cassette was amplified with the primer pair SEQ ID NO:50 and SEQ ID NO:51 using pKD3 as the template. The primer SEQ ID NO:50 contains 80 bp of homology to the 5′ end of pta and 20 bp of homology to the template DNA, pKD3. The primer SEQ ID NO:51 contains 80 bp of homology to the 3′ end of ackA and 20 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of chloramphenicol to give strain MG1655 ΔackA-pta::Cm. The deletion of the pta-ackA genes was confirmed by PCR, using the primer pair SEQ ID NO:52 and SEQ ID NO:53. The wild-type strain gives a 3.8 kb PCR product while the recombinant strain gives a characteristic 1.6 kb PCR product. A P1 phage lysate was prepared and used to pass the mutation to strain Triple 1.6btuR 1.6yqhD to form strain Triple 1.6btuR 1.6yqhD AackA-pta::Cm. The chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to give Triple 1.6btuR 1.6yqhD AackA-pta (renamed TripleTriple (TT)). The Triple 1.6btuR 1.6yqhD and TT strains were electrotransformed with plasmid pSYCO109. [0364]
    • EXAMPLE 15 Measurement of Phosphotransacetylase (Pta) Enzyme Activity in Strain Triple Triple/pSYCO109 Compared to Strain Triple 1.6btuR 1.6vqhD/pSYCO109
  • Fermentations with TT/pSYCO109 and Triple 1.6btuR 1.6yqhD/pSYCO109 were carried out in 14L fermenters as described in the General Methods with the following differences in control parameters for fermentation. A typical fermentation with Triple 1.6btuR 1.6yqhD/pSYCO106 was described in Example 13A. [0365]
  • A pre-culture of TT/pSYCO109 was grown in 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask to an OD550 of approximately 1 AU. A seed fermenter prepared as described was inoculated with 10.8 mL of that culture. After 30.5 hrs of fermentation time, 1.2 L of the culture was transferred to a production fermenter. This fermenter received a single 16 mg bolus of vitamin B[0366] 12 1 hr after inoculation. The final concentration of 1,3-propanediol in a typical fermentation was 114 g/L and the yield was 48%.
  • Samples from a typical Triple 1.6btuR 1.6yghD/pSYCO109 fermentation were analyzed for Pta enzyme activity. A specific activity of 0.4 U/mg protein was obtained. Samples assayed from a typical TT/pSYCO109 fermentation showed no detectable Pta enzyme activity. [0367]
    • EXAMPLE 16 Improved Stability of Molar Yields in Strain TT/pSYCO109 Compared to Triple 1.6btuR 1.6yqhD/pSYCO109
  • Duplicate shake flasks cultures were grown with strains Triple btuR 1.6 yqhD, pSYCO109, and TT pSYCO109. After incubating one colony for 10 hr in LB+50 mg/L spectinomycin, 100 mL of culture were transferred in 30 mL TM2 medium with 2% glucose and with or without 50 ppm spectinomycin (day 1). In order to study the stability of the yield, a 100 mL volume of the day 1 cultures was transferred after 24 hr to a fresh volume of 30 mL TM2 media containing 2% glucose with or without 50 ppm spectinomycin. This was repeated 4 times. The molar yield was calculated as in Example 2 at the end of each 24 hr period and results are given below in Table 9. The ackA-pta deletion stabilizes the molar yield, therefore improving 1,3-propanediol production. [0368]
    TABLE 9
    Molar Yield
    With (+) or Molar Molar Molar Molar Molar
    Without (−) Yield Yield Yield Yield Yield
    Strain Spetinomycin Day 1 Day 2 Day3 Day 4 Day 5
    Triple btuR, 1.6yqhD, + 1.24 1.28 1.06 0.84 0.79
    pSYCO109
    TT, pSYCO109 + 1.15 1.22 1.24 1.24 1.07
    Triple btuR 1.6yqhD, − 1.23 1.04 0.95 0.61 0.25
    pSYCO109
    TT, pSYCO109 − 1.23 1.22 1.23 1.13 1.11
    • EXAMPLE 17 Struction of E. coli Strain with Deletion Mutations in Aldehyde Dehydrogenases by Linear DNA Transformation
  • An aldA deletion [for reference, see GenBank, Accession # U00096] was made by replacing 1.3 kb of the coding region with the FRT-CmR-FRT cassette of pKD3. The cassette was amplified with the primer pair SEQ ID NO:57 and SEQ ID NO:58 using pKD3 as the template. The primer SEQ ID NO:57 contains 80 bp of homology to the 5′ end of aldA and 20 bp of homology to the template DNA, pKD3. The primer SEQ ID NO:58 contains 80 bp of homology to the 3′ end of aldA and 20 bp of homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol. The deletion of the aldA gene was confirmed by PCR, using the primer pair SEQ ID NO:59 and SEQ ID NO:60. The wild-type strain gives a 2.0 kb PCR product while the recombinant strain gives a characteristic 1.8 kb PCR product. A P1 lysate of that strain was prepared and used to move the mutation to the strain TT to form the TT ΔaldA::Cm strain. The chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to create TT aldA. [0369]
  • An aldB deletion [for reference, see GenBank, Accession # U00096] was made by replacing 1.5 kb of the coding region with the FRT-CmR-FRT cassette of the pKD3. A replacement cassette was amplified with the primer pair SEQ ID NO:61 and SEQ ID NO:62 using pKD3 as the template. The primer SEQ ID NO:61 contains 80 bp of homology to the 5′-end of aldB and 20 bp of homology to pKD3. Primer SEQ ID NO:62 contains 80 bp of homology to the 3′ end of aldB and 20 bp homology to pKD3. The PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells. Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol. The deletion of the aldB gene was confirmed by PCR, using the primer pair SEQ ID NO:63 and SEQ ID NO:64. The wild-type strain gives a 1.5 kb PCR product while the recombinant strain gives a characteristic 1.1 kb PCR product. A P1 lysate was prepared and used to move the mutation to the TT strain to form the TT ΔaldB::Cm strain. A chloramphenicol-resistant clone was checked by genomic PCR with the primer pair SEQ ID NO:63 and SEQ ID NO:64 to insure that the mutation was present. [0370]
    • EXAMPLE 17A Production of Glycerol with Strain FMP′ 1.5gap/pSYCO106
  • Strain FMP′ 1.5gapA was transformed with the plasmid pSYCO106. Production of glycerol was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermentation. A thawed frozen vial of FMP′ 1.5gap/pSYCOl06 was transferred to 500 mL SBG1% with 50 mg/L spectinomycin to prepare the pre-culture. No vitamin B[0371] 12 was added to the fermenter. A typical fermentation resulted in the production of 202 g/L glycerol with a molar yield of 115%.
    • EXAMPLE 17B Production of Glycerol with Strain TT/pSYCO109
  • Production of glycerol was determined in 14 L fermenters as described in General Methods with the following differences in control parameters for fermentation. A single colony of TT/pSYCO109 on a LB plate with 50 mg/L spectinomycin was transferred to 30 mL LB with 100 mg/L spectinomycin in a 250 mL flask. When an OD550 of approximately 1 Au was reached, 10.8 mL of the culture was used to inoculate a fermenter prepared as described. No vitamin B[0372] 12 was added to the fermenter. A typical fermentation resulted in the production of 302 g/L glycerol with a molar yield of 137%.
  • 1 68 1 1137 DNA Artificial Sequence partial DNA sequence of plasmid pLoxCat27 comprising the LoxP-Cat-LoxP cassette 1 ctcggatcca ctagtaacgg ccgccagtgt gctggaattc gcccttggcc gcataacttc 60 gtatagtata cattatacga agttatctag agttgcatgc ctgcaggtcc gaatttctgc 120 cattcatccg cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata 180 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 240 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 300 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 360 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 420 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 480 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 540 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 600 ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag 660 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 720 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 780 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 840 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 900 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 960 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt 1020 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggactcta gataacttcg 1080 tatagtatac attatacgaa gttatgaagg gcgaattctg cagatatcca tcacact 1137 2 61 DNA Artificial Sequence Primer ArcA1 2 cacattctta tcgttgaaga cgagttggta acacgcaaca cgtgtaggct ggagctgctt 60 c 61 3 62 DNA Artificial Sequence Primer ArcA2 3 ttccagatca ccgcagaagc gataaccttc accgtgaatg gtcatatgaa tatcctcctt 60 ag 62 4 24 DNA Artificial Sequence Primer ArcA3 4 agttggtaac acgcaacacg caac 24 5 23 DNA Artificial Sequence Primer ArcA4 5 cgcagaagcg ataaccttca ccg 23 6 1320 DNA Artificial Sequence Partial sequence of pLoxCat1 comprising the lox-Cat-loxP cassette 6 aagcttaagg tgcacggccc acgtggccac tagtacttct cgaggtcgac ggtatcgata 60 agctggatcc ataacttcgt ataatgtatg ctatacgaag ttatctagag tccgaataaa 120 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg 180 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca 240 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt 300 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat 360 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata 420 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca 480 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc 540 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg 600 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc 660 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt 720 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca 780 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg 840 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg 900 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg 960 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctggtg 1020 ctacgcctga ataagtgata ataagcggat gaatggcaga aattcggacc tgcaggcatg 1080 caactctaga taacttcgta taatgtatgc tatacgaagt tatgcggccg ccatatgcat 1140 cctaggccta ttaatattcc ggagtatacg tagccggcta acgttctagc atgcgaaatt 1200 taaagcgctg atatcgatcg cgcgcagatc tgtcatgatg atcattgcaa ttggatccat 1260 atatagggcc cggggttata attacctcag gtcgacgtcc catggccatt gaattcgtaa 1320 7 61 DNA Artificial Sequence Primer GalA 7 tcggttttca cagttgttac atttcttttc agtaaagtct ggatgcatat ggcggccgca 60 t 61 8 65 DNA Artificial Sequence Primer GalP2 8 catgatgccc tccaatatgg ttatttttta ttgtgaatta gtctgtttcc tgtgtgaaat 60 tgtta 65 9 60 DNA Artificial Sequence Primer GlkA 9 acttagtttg cccagcttgc aaaaggcatc gctgcaattg gatgcatatg gcggccgcat 60 10 67 DNA Artificial Sequence Primer Glk2 10 cattcttcaa ctgctccgct aaagtcaaaa taattctttc tcgtctgttt cctgtgtgaa 60 attgtta 67 11 1270 DNA Artificial Sequence LoxP-cat-loxP Trc cassette “insert” 11 ggatgcatat ggcggccgca taacttcgta tagcatacat tatacgaagt tatctagagt 60 tgcatgcctg caggtccgaa tttctgccat tcatccgctt attatcactt attcaggcgt 120 agcaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca 180 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac 240 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt 300 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact 360 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg 420 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg 480 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa 540 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat 600 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa 660 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg 720 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg 780 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc 840 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa 900 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt 960 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg 1020 tatttattcg gactctagat aacttcgtat agcatacatt atacgaagtt atggatcatg 1080 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg 1140 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg 1200 acaattaatc atccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 1260 ggaaacagac 1270 12 30 DNA Artificial Sequence Primer GalB1 12 actttggtcg tgaacatttc ccgtgggaaa 30 13 28 DNA Artificial Sequence Primer GalC11 13 agaaagataa gcaccgagga tcccgata 28 14 26 DNA Artificial Sequence Primer GlkB1 14 aacaggagtg ccaaacagtg cgccga 26 15 30 DNA Artificial Sequence Primer GlkC11 15 ctattcggcg caaaatcaac gtgaccgcct 30 16 99 DNA Artificial Sequence Primer edd1 16 atgaatccac aattgttacg cgtaacaaat cgaatcattg aacgttcgcg cgagactcgc 60 tctgcttatc tcgcccggat ttatcgataa gctggatcc 99 17 98 DNA Artificial Sequence Primer edd2 17 ttaaaaagtg atacaggttg cgccctgttc ggcaccggac agtttttcac gcaaggcgct 60 gaataattca cgtcctgtcg gatgcatatg gcggccgc 98 18 22 DNA Artificial Sequence Primer edd3 18 taacatgatc ttgcgcagat tg 22 19 21 DNA Artificial Sequence Primer edd4 19 actgcacact cggtacgcag a 21 20 29 DNA Artificial Sequence CN1, encoding mutated trc promoter driving glk expression 20 ctgacaatta atcatccggc tcgtataat 29 21 29 DNA Artificial Sequence CN2, encoding parent trc promoter 21 ttgacaatta atcatccggc tcgtataat 29 22 25 DNA Artificial Sequence Primer gapA1 22 atgaccatct gaccatttgt gtcaa 25 23 25 DNA Artificial Sequence Primer gapA2 23 aatgcgctaa cagcgtaaag tcgtg 25 24 35 DNA Artificial Sequence Primer gapA3 24 gatacctact ttgatagtca catattccac cagct 35 25 35 DNA Artificial Sequence Primer gapA4 25 agctggtgga atatgtgact atcaaagtag gtatc 35 26 35 DNA Artificial Sequence Primer gapA5 26 gatacctact ttgatagtca aatattccac cagct 35 27 35 DNA Artificial Sequence Primer gapA6 27 agctggtgga atatttgact atcaaagtag gtatc 35 28 42 DNA Artificial Sequence short 1.5 GI promoter 28 gcccttgact atgccacatc ctgagcaaat aattcaacca ct 42 29 98 DNA Artificial Sequence Primer gapA-R1 29 agtcatatat tccaccagct atttgttagt gaataaaagt ggttgaatta tttgctcagg 60 atgtggcata gtcaagggca tatgaatatc ctccttag 98 30 80 DNA Artificial Sequence Primer gapA-R2 30 gctcacatta cgtgactgat tctaacaaaa cattaacacc aactggcaaa attttgtccg 60 tgtaggctgg agctgcttcg 80 31 42 DNA Artificial Sequence short 1.20 GI promoter 31 gcccttgacg atgccacatc ctgagcaaat aattcaacca ct 42 32 42 DNA Artificial Sequence short 1.6 GI promoter 32 gcccttgaca atgccacatc ctgagcaaat aattcaacca ct 42 33 24 DNA Artificial Sequence Primer gapA-R3 33 gtcgacaaac gctggtatac ctca 24 34 98 DNA Artificial Sequence Primer gapA-R4 34 agtcatatat tccaccagct atttgttagt gaataaaagt ggttgaatta tttgctcagg 60 atgtggcatc gtcaagggca tatgaatatc ctccttag 98 35 98 DNA Artificial Sequence Primer gapA-R5 35 agtcatatat tccaccagct atttgttagt gaataaaagt ggttgaatta tttgctcagg 60 atgtggcatt gtcaagggca tatgaatatc ctccttag 98 36 60 DNA Artificial Sequence Primer mgsA-1 36 gtacattatg gaactgacga ctcgcacttt acctgcgcgg tgtaggctgg agctgcttcg 60 37 60 DNA Artificial Sequence Primer mgsA-2 37 cttcagacgg tccgcgagat aacgctgata atcggggatc catatgaata tcctccttag 60 38 22 DNA Artificial Sequence Primer mgsA-3 38 cttgaattgt tggatggcga tg 22 39 21 DNA Artificial Sequence Primer mgsA-4 39 cgtcacgtta ttggatgaga g 21 40 100 DNA Artificial Sequence Primer PppcF 40 cgatttttta acatttccat aagttacgct tatttaaagc gtcgtgaatt taatgacgta 60 aattcctgct atttattcgt gtgtaggctg gagctgcttc 100 41 100 DNA Artificial Sequence Primer PppcR 41 tcgcattggc gcgaatatgc tcgggctttg cttttcgtca gtggttgaat tatttgctca 60 ggatgtggca ttgtcaaggg catatgaata tcctccttag 100 42 30 DNA Artificial Sequence Primer SeqppcR 7 42 gcggaatatt gttcgttcat attaccccag 30 43 90 DNA Artificial Sequence Primer 3G144 43 ccaggctgat tgaaatgccc ttctgtttca ggcataaagc cccaaagtca taaagtacac 60 tggcagcgcg gtgtaggctg gagctgcttc 90 44 93 DNA Artificial Sequence Primer 3G145 44 gcatggctac tcctcaacga cgttgtctgt tagtggttga attatttgct caggatgtgg 60 cattgtcaag ggcattccgg ggatccgtcg acc 93 45 25 DNA Artificial Sequence Primer YCIKUp 45 gataataccg cgttcatcct gggcc 25 46 25 DNA Artificial Sequence Primer YCIKDn 46 gcgagttcac ttcatgggcg tccat 25 47 98 DNA Artificial Sequence Primer yqhCFRTF 47 ttaattcccc tgcatcgccc gcattcttgc cgcatcttcc cccggcgtca caccgaagta 60 acgtttaaac tcacggctgt gtaggctgga gctgcttc 98 48 100 DNA Artificial Sequence Primer CyqhD1.6 48 ctcccttgct gggccaatat gagggcagag aacgatctgc ctggttgaat tatttgctca 60 ggatgtggca ttgtcaaggg catatgaata tcctccttag 100 49 46 DNA Artificial Sequence Primer yqhBF 49 atcgcccgca ttcttgccgc atcttccccc ggcgtcacac cgaagt 46 50 100 DNA Artificial Sequence Primer pta 1 50 atgtcgagta agttagtact ggttctgaac tgcggtagtt cttcactgaa atttgccatc 60 atcgatgcag taaatggtga tgtgtaggct ggagctgctt 100 51 100 DNA Artificial Sequence Primer ack-pta 2 51 ttactgctgc tgtgcagact gaatcgcagt cagcgcgatg gtgtagacga tatcgtcaac 60 cagtgcgcca cgggacaggt catatgaata tcctccttag 100 52 20 DNA Artificial Sequence Primer ack-U 52 attcattgag tcgtcaaatt 20 53 20 DNA Artificial Sequence Primer ack-D 53 attgcggaca tagcgcaaat 20 54 98 DNA Artificial Sequence Primer ptsHFRT1 54 atgttccagc aagaagttac cattaccgct ccgaacggtc tgcacacccg ccctgctgcc 60 cagtttgtaa aagaagctgt gtaggctgga gctgcttc 98 55 97 DNA Artificial Sequence Primer crrFRT11 55 ttacttcttg atgcggataa ccggggtttc acccacggtt acgctaccgg acagtttgat 60 cagttctttg atttcgtcat atgaatatcc tccttag 97 56 36 DNA Artificial Sequence Primer crrR 56 cctgttttgt gctcagctca tcagtggctt gctgaa 36 57 100 DNA Artificial Sequence Primer DaldAF 57 atgtcagtac ccgttcaaca tcctatgtat atcgatggac agtttgttac ctggcgtgga 60 gacgcatgga ttgatgtggt tgtgtaggct ggagctgctt 100 58 100 DNA Artificial Sequence Primer DaldAR 58 ttaagactgt aaataaacca cctgggtctg cagatattca tgcaagccat gtttaccatc 60 tgcgccgcca ataccggatt catatgaata tcctccttag 100 59 20 DNA Artificial Sequence Primer aldAF300 59 ttatcgttca cgttgatttt 20 60 20 DNA Artificial Sequence Primer aldAR300 60 gaaaaaagtg actgccgaag 20 61 100 DNA Artificial Sequence Primer DaldBF 61 cgtctaccct tgttatacct cacaccgcaa ggagacgatc atgaccaata atcccccttc 60 agcacagatt aagcccggcg gtgtaggctg gagctgcttc 100 62 100 DNA Artificial Sequence Primer DaldBR 62 gcatcaggca atgaataccc aatgcgacca gcttcttata tcagaacagc cccaacggtt 60 tatccgagta gctcaccagc catatgaata tcctccttag 100 63 22 DNA Artificial Sequence Primer aldBF 63 atgaccaata atcccccttc ag 22 64 21 DNA Artificial Sequence Primer aldBR 64 gcttcttata tcagaacagc c 21 65 13669 DNA Artificial sequence Plasmid pSYCO101 65 tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60 taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120 acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180 ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240 gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660 atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020 gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260 cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320 atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380 ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440 gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500 ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560 atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620 atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680 atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740 gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800 ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860 gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920 gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980 tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040 ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100 cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160 ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220 ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280 ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340 ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400 aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460 cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520 atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580 tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640 taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700 tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760 gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820 tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880 aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940 gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000 gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060 atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120 gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180 tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240 atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300 tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360 gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420 ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480 aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540 aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600 ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660 caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720 agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780 cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840 tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900 acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960 tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020 cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080 cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140 gcgagccgtc acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200 atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260 caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320 gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380 cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440 tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500 caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560 ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620 gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680 gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740 ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800 atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860 ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920 cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980 agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040 gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100 gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160 gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220 cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280 atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340 cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400 gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460 ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520 tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580 ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640 tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700 tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760 tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga caatgccaca 5820 tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880 ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940 gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000 acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060 acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120 acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180 tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240 aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300 tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360 tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420 cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480 gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540 gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600 ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660 gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720 agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780 ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840 gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900 ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960 agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020 acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080 gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140 gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200 ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260 acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320 atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380 tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440 tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500 gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560 ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620 tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680 gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740 cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800 gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860 cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920 tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980 ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040 tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100 aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160 gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220 cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280 gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340 ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400 ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460 ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520 gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580 gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640 cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700 cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760 cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820 tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880 tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940 cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000 ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060 tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120 gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180 gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240 gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300 ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360 cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420 aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480 cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540 tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600 ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660 cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720 aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780 cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840 ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900 gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960 gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020 ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080 cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140 taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200 tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260 cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320 ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380 gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440 tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500 tctagagtac tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 10560 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccggggc 10620 ggccgcgcta gcgcccgatc cagctggagt ttgtagaaac gcaaaaaggc catccgtcag 10680 gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc 10740 tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag 10800 agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg 10860 ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg 10920 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc 10980 cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc tgtatcaggc 11040 tgaaaatctt ctctcatccg ccaaaacagc caagcttgca tgcctgcagc ccgggttacc 11100 atttcaacag atcgtcctta gcatataagt agtcgtcaaa aatgaattca acttcgtctg 11160 tttcggcatt gtagccgcca actctgatgg attcgtggtt tttgacaatg atgtcacagc 11220 ctttttcctt taggaagtcc aagtcgaaag tagtggcaat accaatgatc ttacaaccgg 11280 cggcttttcc ggcggcaata cctgctggag cgtcttcaaa tactactacc ttagatttgg 11340 aagggtcttg ctcattgatc ggatatccta agccattcct gcccttcaga tatggttctg 11400 gatgaggctt accctgtttg acatcattag cggtaatgaa gtactttggt ctcctgattc 11460 ccagatgctc gaaccatttt tgtgccatat cacgggtacc ggaagttgcc acagcccatt 11520 tctcttttgg tagagcgttc aaagcgttgc acagcttaac tgcacctggg acttcaatgg 11580 atttttcacc gtacttgacc ggaatttcag cttctaattt gttaacatac tcttcattgg 11640 caaagtctgg agcgaactta gcaatggcat caaacgttct ccaaccatgc gagacttgga 11700 taacgtgttc agcatcgaaa taaggtttgt ccttaccgaa atccctccag aatgcagcaa 11760 tggctggttg agagatgata atggtaccgt cgacgtcgaa caaagcggcg ttaactttca 11820 aagatagagg tttagtagtc aatcccataa ttctagtctg tttcctggat ccaataaatc 11880 taatcttcat gtagatctaa ttcttcaatc atgtccggca ggttcttcat tgggtagttg 11940 ttgtaaacga tttggtatac ggcttcaaat aatgggaagt cttcgacaga gccacatgtt 12000 tccaaccatt cgtgaacttc tttgcaggta attaaacctt gagcggattg gccattcaac 12060 aactcctttt cacattccca ggcgtcctta ccagaagtag ccattagcct agcaaccttg 12120 acgtttctac caccagcgca ggtggtgatc aaatcagcaa caccagcaga ctcttggtag 12180 tatgtttctt ctctagattc tgggaaaaac atttgaccga atctgatgat ctcacccaaa 12240 ccgactcttt ggatggcagc agaagcgttg ttaccccagc ctagaccttc gacgaaacca 12300 caacctaagg caacaacgtt cttcaaagca ccacagatgg agataccagc aacatcttcg 12360 atgacactaa cgtggaagta aggtctgtgg aacaaggcct ttagaacctt atggtcgacg 12420 tccttgccct cgcctctgaa atcctttgga atgtggtaag caactgttgt ttcagaccag 12480 tgttcttgag cgacttcggt ggcaatgtta gcaccagata gagcaccaca ttgaatacct 12540 agttcctcag tgatgtaaga ggatagcaat tggacacctt tagcaccaac ttcaaaaccc 12600 tttagacagg agatagctct gacgtgtgaa tcaacatgac ctttcaattg gctacagata 12660 cggggcaaaa attgatgtgg aatgttgaaa acgatgatgt cgacatcctt gactgaatca 12720 atcaagtctg gattagcaac caaattgtcg ggtagagtga tgccaggcaa gtatttcacg 12780 ttttgatgtc tagtatttat gatttcagtc aatttttcac cattgatctc ttcttcgaac 12840 acccacattt gtactattgg agcgaaaact tctgggtatc ccttacaatt ttcggcaacc 12900 accttggcaa tagtagtacc ccagttacca gatccaatca cagtaacctt gaaaggcttt 12960 tcggcagcct tcaaagaaac agaagaggaa cttctctttc taccagcatt caagtggccg 13020 gaagttaagt ttaatctatc agcagcagca gccatggaat tgtcctcctt actagtcatg 13080 gtctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccggat 13140 gattaattgt caacagctca tttcagaata tttgccagaa ccgttatgat gtcggcgcaa 13200 aaaacattat ccagaacggg agtgcgcctt gagcgacacg aattatgcag tgatttacga 13260 cctgcacagc cataccacag cttccgatgg ctgcctgacg ccagaagcat tggtgcacgc 13320 tagccagtac atttaaatgg taccctctag tcaaggcctt aagtgagtcg tattacggac 13380 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 13440 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 13500 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta 13560 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 13620 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgagct 13669 66 13543 DNA artificial sequence Plasmid pSYCO103 66 tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60 taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120 acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180 ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240 gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660 atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020 gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260 cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320 atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380 ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440 gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500 ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560 atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620 atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680 atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740 gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800 ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860 gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920 gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980 tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040 ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100 cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160 ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220 ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280 ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340 ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400 aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460 cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520 atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580 tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640 taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700 tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760 gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820 tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880 aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940 gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000 gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060 atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120 gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180 tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240 atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300 tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360 gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420 ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480 aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540 aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600 ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660 caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720 agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780 cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840 tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900 acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960 tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020 cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080 cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140 gcgagccgtc acgcccttga ctatgccaca tcctgagcaa ataattcaac cactaaacaa 4200 atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260 caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320 gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380 cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440 tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500 caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560 ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620 gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680 gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740 ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800 atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860 ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920 cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980 agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040 gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100 gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160 gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220 cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280 atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340 cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400 gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460 ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520 tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580 ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640 tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700 tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760 tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga ctatgccaca 5820 tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880 ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940 gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000 acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060 acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120 acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180 tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240 aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300 tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360 tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420 cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480 gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540 gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600 ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660 gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720 agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780 ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840 gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900 ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960 agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020 acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080 gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140 gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200 ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260 acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320 atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380 tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440 tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500 gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560 ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620 tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680 gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740 cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800 gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860 cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920 tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980 ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040 tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100 aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160 gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220 cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280 gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340 ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400 ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460 ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520 gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580 gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640 cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700 cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760 cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820 tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880 tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940 cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000 ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060 tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120 gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180 gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240 gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300 ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360 cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420 aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480 cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540 tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600 ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660 cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720 aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780 cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840 ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900 gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960 gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020 ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080 cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140 taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200 tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260 cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320 ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380 gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440 tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500 tctagcgtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 10560 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 10620 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 10680 aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 10740 agaccatgac tagtaaggag gacaattcca tggctgctgc tgctgataga ttaaacttaa 10800 cttccggcca cttgaatgct ggtagaaaga gaagttcctc ttctgtttct ttgaaggctg 10860 ccgaaaagcc tttcaaggtt actgtgattg gatctggtaa ctggggtact actattgcca 10920 aggtggttgc cgaaaattgt aagggatacc cagaagtttt cgctccaata gtacaaatgt 10980 gggtgttcga agaagagatc aatggtgaaa aattgactga aatcataaat actagacatc 11040 aaaacgtgaa atacttgcct ggcatcactc tacccgacaa tttggttgct aatccagact 11100 tgattgattc agtcaaggat gtcgacatca tcgttttcaa cattccacat caatttttgc 11160 cccgtatctg tagccaattg aaaggtcatg ttgattcaca cgtcagagct atctcctgtc 11220 taaagggttt tgaagttggt gctaaaggtg tccaattgct atcctcttac atcactgagg 11280 aactaggtat tcaatgtggt gctctatctg gtgctaacat tgccaccgaa gtcgctcaag 11340 aacactggtc tgaaacaaca gttgcttacc acattccaaa ggatttcaga ggcgagggca 11400 aggacgtcga ccataaggtt ctaaaggcct tgttccacag accttacttc cacgttagtg 11460 tcatcgaaga tgttgctggt atctccatct gtggtgcttt gaagaacgtt gttgccttag 11520 gttgtggttt cgtcgaaggt ctaggctggg gtaacaacgc ttctgctgcc atccaaagag 11580 tcggtttggg tgagatcatc agattcggtc aaatgttttt cccagaatct agagaagaaa 11640 catactacca agagtctgct ggtgttgctg atttgatcac cacctgcgct ggtggtagaa 11700 acgtcaaggt tgctaggcta atggctactt ctggtaagga cgcctgggaa tgtgaaaagg 11760 agttgttgaa tggccaatcc gctcaaggtt taattacctg caaagaagtt cacgaatggt 11820 tggaaacatg tggctctgtc gaagacttcc cattatttga agccgtatac caaatcgttt 11880 acaacaacta cccaatgaag aacctgccgg acatgattga agaattagat ctacatgaag 11940 attagattta ttggatccag gaaacagact agaattatgg gattgactac taaacctcta 12000 tctttgaaag ttaacgccgc tttgttcgac gtcgacggta ccattatcat ctctcaacca 12060 gccattgctg cattctggag ggatttcggt aaggacaaac cttatttcga tgctgaacac 12120 gttatccaag tctcgcatgg ttggagaacg tttgatgcca ttgctaagtt cgctccagac 12180 tttgccaatg aagagtatgt taacaaatta gaagctgaaa ttccggtcaa gtacggtgaa 12240 aaatccattg aagtcccagg tgcagttaag ctgtgcaacg ctttgaacgc tctaccaaaa 12300 gagaaatggg ctgtggcaac ttccggtacc cgtgatatgg cacaaaaatg gttcgagcat 12360 ctgggaatca ggagaccaaa gtacttcatt accgctaatg atgtcaaaca gggtaagcct 12420 catccagaac catatctgaa gggcaggaat ggcttaggat atccgatcaa tgagcaagac 12480 ccttccaaat ctaaggtagt agtatttgaa gacgctccag caggtattgc cgccggaaaa 12540 gccgccggtt gtaagatcat tggtattgcc actactttcg acttggactt cctaaaggaa 12600 aaaggctgtg acatcattgt caaaaaccac gaatccatca gagttggcgg ctacaatgcc 12660 gaaacagacg aagttgaatt catttttgac gactacttat atgctaagga cgatctgttg 12720 aaatggtaac ccgggctgca ggcatgcaag cttggctgtt ttggcggatg agagaagatt 12780 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct 12840 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt 12900 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat 12960 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 13020 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 13080 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc 13140 catcctgacg gatggccttt ttgcgtttct acaaactcca gctggatcgg gcgctagagt 13200 atacatttaa atggtaccct ctagtcaagg ccttaagtga gtcgtattac ggactggccg 13260 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 13320 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 13380 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 13440 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 13500 agttaagcca gccccgacac ccgccaacac ccgctgacga gct 13543 67 13543 DNA Artificial sequence Plasmid pSYCO106 67 tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60 taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120 acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180 ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240 gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660 atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020 gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260 cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320 atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380 ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440 gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500 ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560 atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620 atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680 atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740 gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800 ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860 gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920 gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980 tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040 ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100 cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160 ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220 ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280 ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340 ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400 aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460 cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520 atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580 tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640 taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700 tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760 gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820 tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880 aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940 gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000 gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060 atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120 gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180 tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240 atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300 tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360 gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420 ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480 aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540 aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600 ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660 caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720 agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780 cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840 tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900 acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960 tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020 cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080 cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140 gcgagccgtc acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200 atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260 caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320 gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380 cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440 tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500 caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560 ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620 gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680 gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740 ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800 atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860 ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920 cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980 agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040 gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100 gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160 gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220 cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280 atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340 cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400 gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460 ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520 tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580 ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640 tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700 tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760 tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga caatgccaca 5820 tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880 ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940 gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000 acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060 acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120 acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180 tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240 aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300 tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360 tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420 cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480 gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540 gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600 ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660 gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720 agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780 ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840 gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900 ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960 agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020 acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080 gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140 gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200 ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260 acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320 atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380 tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440 tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500 gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560 ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620 tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680 gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740 cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800 gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860 cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920 tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980 ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040 tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100 aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160 gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220 cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280 gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340 ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400 ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460 ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520 gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580 gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640 cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700 cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760 cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820 tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880 tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940 cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000 ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060 tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120 gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180 gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240 gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300 ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360 cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420 aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480 cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540 tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600 ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660 cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720 aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780 cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840 ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900 gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960 gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020 ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080 cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140 taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200 tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260 cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320 ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380 gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440 tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500 tctagcgtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 10560 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 10620 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 10680 aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 10740 agaccatgac tagtaaggag gacaattcca tggctgctgc tgctgataga ttaaacttaa 10800 cttccggcca cttgaatgct ggtagaaaga gaagttcctc ttctgtttct ttgaaggctg 10860 ccgaaaagcc tttcaaggtt actgtgattg gatctggtaa ctggggtact actattgcca 10920 aggtggttgc cgaaaattgt aagggatacc cagaagtttt cgctccaata gtacaaatgt 10980 gggtgttcga agaagagatc aatggtgaaa aattgactga aatcataaat actagacatc 11040 aaaacgtgaa atacttgcct ggcatcactc tacccgacaa tttggttgct aatccagact 11100 tgattgattc agtcaaggat gtcgacatca tcgttttcaa cattccacat caatttttgc 11160 cccgtatctg tagccaattg aaaggtcatg ttgattcaca cgtcagagct atctcctgtc 11220 taaagggttt tgaagttggt gctaaaggtg tccaattgct atcctcttac atcactgagg 11280 aactaggtat tcaatgtggt gctctatctg gtgctaacat tgccaccgaa gtcgctcaag 11340 aacactggtc tgaaacaaca gttgcttacc acattccaaa ggatttcaga ggcgagggca 11400 aggacgtcga ccataaggtt ctaaaggcct tgttccacag accttacttc cacgttagtg 11460 tcatcgaaga tgttgctggt atctccatct gtggtgcttt gaagaacgtt gttgccttag 11520 gttgtggttt cgtcgaaggt ctaggctggg gtaacaacgc ttctgctgcc atccaaagag 11580 tcggtttggg tgagatcatc agattcggtc aaatgttttt cccagaatct agagaagaaa 11640 catactacca agagtctgct ggtgttgctg atttgatcac cacctgcgct ggtggtagaa 11700 acgtcaaggt tgctaggcta atggctactt ctggtaagga cgcctgggaa tgtgaaaagg 11760 agttgttgaa tggccaatcc gctcaaggtt taattacctg caaagaagtt cacgaatggt 11820 tggaaacatg tggctctgtc gaagacttcc cattatttga agccgtatac caaatcgttt 11880 acaacaacta cccaatgaag aacctgccgg acatgattga agaattagat ctacatgaag 11940 attagattta ttggatccag gaaacagact agaattatgg gattgactac taaacctcta 12000 tctttgaaag ttaacgccgc tttgttcgac gtcgacggta ccattatcat ctctcaacca 12060 gccattgctg cattctggag ggatttcggt aaggacaaac cttatttcga tgctgaacac 12120 gttatccaag tctcgcatgg ttggagaacg tttgatgcca ttgctaagtt cgctccagac 12180 tttgccaatg aagagtatgt taacaaatta gaagctgaaa ttccggtcaa gtacggtgaa 12240 aaatccattg aagtcccagg tgcagttaag ctgtgcaacg ctttgaacgc tctaccaaaa 12300 gagaaatggg ctgtggcaac ttccggtacc cgtgatatgg cacaaaaatg gttcgagcat 12360 ctgggaatca ggagaccaaa gtacttcatt accgctaatg atgtcaaaca gggtaagcct 12420 catccagaac catatctgaa gggcaggaat ggcttaggat atccgatcaa tgagcaagac 12480 ccttccaaat ctaaggtagt agtatttgaa gacgctccag caggtattgc cgccggaaaa 12540 gccgccggtt gtaagatcat tggtattgcc actactttcg acttggactt cctaaaggaa 12600 aaaggctgtg acatcattgt caaaaaccac gaatccatca gagttggcgg ctacaatgcc 12660 gaaacagacg aagttgaatt catttttgac gactacttat atgctaagga cgatctgttg 12720 aaatggtaac ccgggctgca ggcatgcaag cttggctgtt ttggcggatg agagaagatt 12780 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct 12840 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt 12900 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat 12960 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 13020 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 13080 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc 13140 catcctgacg gatggccttt ttgcgtttct acaaactcca gctggatcgg gcgctagagt 13200 atacatttaa atggtaccct ctagtcaagg ccttaagtga gtcgtattac ggactggccg 13260 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 13320 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 13380 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 13440 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 13500 agttaagcca gccccgacac ccgccaacac ccgctgacga gct 13543 68 13402 DNA Artificial sequence Plasmid pSYCO109 68 tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60 taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120 acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180 ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240 gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660 atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020 gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260 cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320 atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380 ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440 gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500 ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560 atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620 atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680 atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740 gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800 ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860 gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920 gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980 tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040 ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100 cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160 ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220 ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280 ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340 ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400 aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460 cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520 atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580 tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640 taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700 tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760 gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820 tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880 aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940 gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000 gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060 atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120 gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180 tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240 atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300 tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360 gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420 ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480 aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540 aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600 ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660 caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720 agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780 cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840 tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900 acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960 tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020 cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080 cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140 gcgagccgtc acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200 atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260 caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320 gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380 cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440 tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500 caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560 ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620 gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680 gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740 ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800 atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860 ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920 cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980 agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040 gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100 gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160 gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220 cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280 atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340 cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400 gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460 ctgcccagct gcaccctgat gcttgcgctt gaactggcct agcaaacaca gaaaaaagcc 5520 cgcacctgac agtgcgggct ttttttttcc taggcgatct gtgctgtttg ccacggtatg 5580 cagcaccagc gcgagattat gggctcgcac gctcgactgt cggacggggg cactggaacg 5640 agaagtcagg cgagccgtca cgcccttgac aatgccacat cctgagcaaa taattcaacc 5700 actaaacaaa tcaaccgcgt ttcccggagg taaccaagct tcaccttttg agccgatgaa 5760 caatgaaaag atcaaaacga tttgcagtac tggcccagcg ccccgtcaat caggacgggc 5820 tgattggcga gtggcctgaa gaggggctga tcgccatgga cagccccttt gacccggtct 5880 cttcagtaaa agtggacaac ggtctgatcg tcgaactgga cggcaaacgc cgggaccagt 5940 ttgacatgat cgaccgattt atcgccgatt acgcgatcaa cgttgagcgc acagagcagg 6000 caatgcgcct ggaggcggtg gaaatagccc gtatgctggt ggatattcac gtcagccggg 6060 aggagatcat tgccatcact accgccatca cgccggccaa agcggtcgag gtgatggcgc 6120 agatgaacgt ggtggagatg atgatggcgc tgcagaagat gcgtgcccgc cggaccccct 6180 ccaaccagtg ccacgtcacc aatctcaaag ataatccggt gcagattgcc gctgacgccg 6240 ccgaggccgg gatccgcggc ttctcagaac aggagaccac ggtcggtatc gcgcgctacg 6300 cgccgtttaa cgccctggcg ctgttggtcg gttcgcagtg cggccgcccc ggcgtgttga 6360 cgcagtgctc ggtggaagag gccaccgagc tggagctggg catgcgtggc ttaaccagct 6420 acgccgagac ggtgtcggtc tacggcaccg aagcggtatt taccgacggc gatgatacgc 6480 cgtggtcaaa ggcgttcctc gcctcggcct acgcctcccg cgggttgaaa atgcgctaca 6540 cctccggcac cggatccgaa gcgctgatgg gctattcgga gagcaagtcg atgctctacc 6600 tcgaatcgcg ctgcatcttc attactaaag gcgccggggt tcagggactg caaaacggcg 6660 cggtgagctg tatcggcatg accggcgctg tgccgtcggg cattcgggcg gtgctggcgg 6720 aaaacctgat cgcctctatg ctcgacctcg aagtggcgtc cgccaacgac cagactttct 6780 cccactcgga tattcgccgc accgcgcgca ccctgatgca gatgctgccg ggcaccgact 6840 ttattttctc cggctacagc gcggtgccga actacgacaa catgttcgcc ggctcgaact 6900 tcgatgcgga agattttgat gattacaaca tcctgcagcg tgacctgatg gttgacggcg 6960 gcctgcgtcc ggtgaccgag gcggaaacca ttgccattcg ccagaaagcg gcgcgggcga 7020 tccaggcggt tttccgcgag ctggggctgc cgccaatcgc cgacgaggag gtggaggccg 7080 ccacctacgc gcacggcagc aacgagatgc cgccgcgtaa cgtggtggag gatctgagtg 7140 cggtggaaga gatgatgaag cgcaacatca ccggcctcga tattgtcggc gcgctgagcc 7200 gcagcggctt tgaggatatc gccagcaata ttctcaatat gctgcgccag cgggtcaccg 7260 gcgattacct gcagacctcg gccattctcg atcggcagtt cgaggtggtg agtgcggtca 7320 acgacatcaa tgactatcag gggccgggca ccggctatcg catctctgcc gaacgctggg 7380 cggagatcaa aaatattccg ggcgtggttc agcccgacac cattgaataa ggcggtattc 7440 ctgtgcaaca gacaacccaa attcagccct cttttaccct gaaaacccgc gagggcgggg 7500 tagcttctgc cgatgaacgc gccgatgaag tggtgatcgg cgtcggccct gccttcgata 7560 aacaccagca tcacactctg atcgatatgc cccatggcgc gatcctcaaa gagctgattg 7620 ccggggtgga agaagagggg cttcacgccc gggtggtgcg cattctgcgc acgtccgacg 7680 tctcctttat ggcctgggat gcggccaacc tgagcggctc ggggatcggc atcggtatcc 7740 agtcgaaggg gaccacggtc atccatcagc gcgatctgct gccgctcagc aacctggagc 7800 tgttctccca ggcgccgctg ctgacgctgg agacctaccg gcagattggc aaaaacgctg 7860 cgcgctatgc gcgcaaagag tcaccttcgc cggtgccggt ggtgaacgat cagatggtgc 7920 ggccgaaatt tatggccaaa gccgcgctat ttcatatcaa agagaccaaa catgtggtgc 7980 aggacgccga gcccgtcacc ctgcacatcg acttagtaag ggagtgacca tgagcgagaa 8040 aaccatgcgc gtgcaggatt atccgttagc cacccgctgc ccggagcata tcctgacgcc 8100 taccggcaaa ccattgaccg atattaccct cgagaaggtg ctctctggcg aggtgggccc 8160 gcaggatgtg cggatctccc gccagaccct tgagtaccag gcgcagattg ccgagcagat 8220 gcagcgccat gcggtggcgc gcaatttccg ccgcgcggcg gagcttatcg ccattcctga 8280 cgagcgcatt ctggctatct ataacgcgct gcgcccgttc cgctcctcgc aggcggagct 8340 gctggcgatc gccgacgagc tggagcacac ctggcatgcg acagtgaatg ccgcctttgt 8400 ccgggagtcg gcggaagtgt atcagcagcg gcataagctg cgtaaaggaa gctaagcgga 8460 ggtcagcatg ccgttaatag ccgggattga tatcggcaac gccaccaccg aggtggcgct 8520 ggcgtccgac tacccgcagg cgagggcgtt tgttgccagc gggatcgtcg cgacgacggg 8580 catgaaaggg acgcgggaca atatcgccgg gaccctcgcc gcgctggagc aggccctggc 8640 gaaaacaccg tggtcgatga gcgatgtctc tcgcatctat cttaacgaag ccgcgccggt 8700 gattggcgat gtggcgatgg agaccatcac cgagaccatt atcaccgaat cgaccatgat 8760 cggtcataac ccgcagacgc cgggcggggt gggcgttggc gtggggacga ctatcgccct 8820 cgggcggctg gcgacgctgc cggcggcgca gtatgccgag gggtggatcg tactgattga 8880 cgacgccgtc gatttccttg acgccgtgtg gtggctcaat gaggcgctcg accgggggat 8940 caacgtggtg gcggcgatcc tcaaaaagga cgacggcgtg ctggtgaaca accgcctgcg 9000 taaaaccctg ccggtggtgg atgaagtgac gctgctggag caggtccccg agggggtaat 9060 ggcggcggtg gaagtggccg cgccgggcca ggtggtgcgg atcctgtcga atccctacgg 9120 gatcgccacc ttcttcgggc taagcccgga agagacccag gccatcgtcc ccatcgcccg 9180 cgccctgatt ggcaaccgtt ccgcggtggt gctcaagacc ccgcaggggg atgtgcagtc 9240 gcgggtgatc ccggcgggca acctctacat tagcggcgaa aagcgccgcg gagaggccga 9300 tgtcgccgag ggcgcggaag ccatcatgca ggcgatgagc gcctgcgctc cggtacgcga 9360 catccgcggc gaaccgggca cccacgccgg cggcatgctt gagcgggtgc gcaaggtaat 9420 ggcgtccctg accggccatg agatgagcgc gatatacatc caggatctgc tggcggtgga 9480 tacgtttatt ccgcgcaagg tgcagggcgg gatggccggc gagtgcgcca tggagaatgc 9540 cgtcgggatg gcggcgatgg tgaaagcgga tcgtctgcaa atgcaggtta tcgcccgcga 9600 actgagcgcc cgactgcaga ccgaggtggt ggtgggcggc gtggaggcca acatggccat 9660 cgccggggcg ttaaccactc ccggctgtgc ggcgccgctg gcgatcctcg acctcggcgc 9720 cggctcgacg gatgcggcga tcgtcaacgc ggaggggcag ataacggcgg tccatctcgc 9780 cggggcgggg aatatggtca gcctgttgat taaaaccgag ctgggcctcg aggatctttc 9840 gctggcggaa gcgataaaaa aatacccgct ggccaaagtg gaaagcctgt tcagtattcg 9900 tcacgagaat ggcgcggtgg agttctttcg ggaagccctc agcccggcgg tgttcgccaa 9960 agtggtgtac atcaaggagg gcgaactggt gccgatcgat aacgccagcc cgctggaaaa 10020 aattcgtctc gtgcgccggc aggcgaaaga gaaagtgttt gtcaccaact gcctgcgcgc 10080 gctgcgccag gtctcacccg gcggttccat tcgcgatatc gcctttgtgg tgctggtggg 10140 cggctcatcg ctggactttg agatcccgca gcttatcacg gaagccttgt cgcactatgg 10200 cgtggtcgcc gggcagggca atattcgggg aacagaaggg ccgcgcaatg cggtcgccac 10260 cgggctgcta ctggccggtc aggcgaatta aacgggcgct cgcgccagcc tctaggtaca 10320 aataaaaaag gcacgtcaga tgacgtgcct tttttcttgt ctagcgtgca ccaatgcttc 10380 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata 10440 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa 10500 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatccggc tcgtataatg 10560 tgtggaattg tgagcggata acaatttcac acaggaaaca gaccatgact agtaaggagg 10620 acaattccat ggctgctgct gctgatagat taaacttaac ttccggccac ttgaatgctg 10680 gtagaaagag aagttcctct tctgtttctt tgaaggctgc cgaaaagcct ttcaaggtta 10740 ctgtgattgg atctggtaac tggggtacta ctattgccaa ggtggttgcc gaaaattgta 10800 agggataccc agaagttttc gctccaatag tacaaatgtg ggtgttcgaa gaagagatca 10860 atggtgaaaa attgactgaa atcataaata ctagacatca aaacgtgaaa tacttgcctg 10920 gcatcactct acccgacaat ttggttgcta atccagactt gattgattca gtcaaggatg 10980 tcgacatcat cgttttcaac attccacatc aatttttgcc ccgtatctgt agccaattga 11040 aaggtcatgt tgattcacac gtcagagcta tctcctgtct aaagggtttt gaagttggtg 11100 ctaaaggtgt ccaattgcta tcctcttaca tcactgagga actaggtatt caatgtggtg 11160 ctctatctgg tgctaacatt gccaccgaag tcgctcaaga acactggtct gaaacaacag 11220 ttgcttacca cattccaaag gatttcagag gcgagggcaa ggacgtcgac cataaggttc 11280 taaaggcctt gttccacaga ccttacttcc acgttagtgt catcgaagat gttgctggta 11340 tctccatctg tggtgctttg aagaacgttg ttgccttagg ttgtggtttc gtcgaaggtc 11400 taggctgggg taacaacgct tctgctgcca tccaaagagt cggtttgggt gagatcatca 11460 gattcggtca aatgtttttc ccagaatcta gagaagaaac atactaccaa gagtctgctg 11520 gtgttgctga tttgatcacc acctgcgctg gtggtagaaa cgtcaaggtt gctaggctaa 11580 tggctacttc tggtaaggac gcctgggaat gtgaaaagga gttgttgaat ggccaatccg 11640 ctcaaggttt aattacctgc aaagaagttc acgaatggtt ggaaacatgt ggctctgtcg 11700 aagacttccc attatttgaa gccgtatacc aaatcgttta caacaactac ccaatgaaga 11760 acctgccgga catgattgaa gaattagatc tacatgaaga ttagatttat tggatccagg 11820 aaacagacta gaattatggg attgactact aaacctctat ctttgaaagt taacgccgct 11880 ttgttcgacg tcgacggtac cattatcatc tctcaaccag ccattgctgc attctggagg 11940 gatttcggta aggacaaacc ttatttcgat gctgaacacg ttatccaagt ctcgcatggt 12000 tggagaacgt ttgatgccat tgctaagttc gctccagact ttgccaatga agagtatgtt 12060 aacaaattag aagctgaaat tccggtcaag tacggtgaaa aatccattga agtcccaggt 12120 gcagttaagc tgtgcaacgc tttgaacgct ctaccaaaag agaaatgggc tgtggcaact 12180 tccggtaccc gtgatatggc acaaaaatgg ttcgagcatc tgggaatcag gagaccaaag 12240 tacttcatta ccgctaatga tgtcaaacag ggtaagcctc atccagaacc atatctgaag 12300 ggcaggaatg gcttaggata tccgatcaat gagcaagacc cttccaaatc taaggtagta 12360 gtatttgaag acgctccagc aggtattgcc gccggaaaag ccgccggttg taagatcatt 12420 ggtattgcca ctactttcga cttggacttc ctaaaggaaa aaggctgtga catcattgtc 12480 aaaaaccacg aatccatcag agttggcggc tacaatgccg aaacagacga agttgaattc 12540 atttttgacg actacttata tgctaaggac gatctgttga aatggtaacc cgggctgcag 12600 gcatgcaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa 12660 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc 12720 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg 12780 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa 12840 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa 12900 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg 12960 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt 13020 tgcgtttcta caaactccag ctggatcggg cgctagagta tacatttaaa tggtaccctc 13080 tagtcaaggc cttaagtgag tcgtattacg gactggccgt cgttttacaa cgtcgtgact 13140 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 13200 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 13260 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 13320 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 13380 cgccaacacc cgctgacgag ct 13402

Claims (8)

What is claimed is:
1. An E. coli strain comprising:
a) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system preventing expression of active PEP-glucose phosphotransferase system proteins;
b) an up regulated endogenous galP gene encoding active galactose-proton symporter;
c) an up regulated endogenous glk gene encoding active glucokinase; and
d) a down regulated endogenous gapA gene encoding active glyceraldehyde 3-phosphate dehydrogenase.
2. The E. coli strain of claim 1, wherein the disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprises one or more of:
i) disrupted endogenous ptsH gene preventing expression of active phosphocarrier protein;
ii) disrupted endogenous ptsl gene preventing expression of active phosphoenolpyruvate-protein phosphotransferase; and
iii) disrupted endogenous crr gene preventing expression of active glucose-specific IIA component.
3. The E. coli strain of claims 1 or 2, further comprising one or more of:
e) a disrupted endogenous arcA gene preventing expression of active aerobic respiration control protein;
f) an up regulated endogenous ppc gene encoding active phosphoenolpyruvate carboxylase;
g) an up regulated endogenous btuR gene encoding active cob(I)alamin adenosyltransferase; and
h) an up regulated yqhD gene encoding active alcohol dehydrogenase.
4. The E. coli strain of claims 1, 2, or 3, further comprising one or more of:
i) a disrupted endogenous mgsA gene preventing the expression of active methylglyoxal synthase;
j) a disrupted endogenous ackA gene preventing the expression of active acetate kinase;
k) a disrupted endogenous pta gene preventing the expression of active phosphotrasacetylase;
l) a disrupted endogenous aldA gene preventing the expression of active aldehyde dehydrogenase A; and
m) a disrupted endogenous aldB gene preventing the expression of active aldehyde dehydrogenase B.
5. The E. coli strain of claims 1, 2, 3, or 4, further comprising one or more of:
n) a disrupted endogenous edd gene preventing expression of active phosphogluconate dehydratase;
o) a disrupted endogenous glpk gene preventing expression of active glycerol kinase; and
p) a disrupted endogenous gldA gene preventing expression of active NADH-dependent glycerol dehydrogenase.
6. A method for the bioproduction of 1,3-propanediol comprising contacting the E. coli strain of claims 1, 2, 3, 4 or 5 with a suitable carbon substrate under suitable conditions.
7. The method of claim 6, wherein the E. coli strain further comprises:
(i) glycerol-3-phosphate dehydrogenase;
(ii) glycerol-3-phosphatase;
(iii) dehydratase; and
(iv) dehydratase reactivation factor.
8. An E. coli strain comprising
a) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system preventing expression of active PEP-glucose phosphotransferase system proteins;
b) an up regulated endogenous galP gene encoding active galactose-proton symporter;
c) an up regulated endogenous glk gene encoding active glucokinase;
d) a down regulated endogenous gapA gene encoding active glyceraldehyde 3-phosphate dehydrogenase.
e) a disrupted endogenous arcA gene preventing expression of active aerobic respiration control protein;
f) an up regulated endogenous ppc gene encoding active phosphoenolpyruvate carboxylase;
g) an up regulated endogenous btuR gene encoding active cob(I)alamin adenosyltransferase;
h) an up regulated yqhD gene encoding active alcohol dehydrogenase.
i) a disrupted endogenous mgsA gene preventing the expression of active methylglyoxal synthase;
j) a disrupted endogenous ackA gene preventing the expression of active acetate kinase;
k) a disrupted endogenous pta gene preventing the expression of active phosphotrasacetylase;
l) a disrupted endogenous aldA gene preventing the expression of active aldehyde dehydrogenase A;
m) a disrupted endogenous aldB gene preventing the expression of active aldehyde dehydrogenase B.
n) a disrupted endogenous edd gene preventing expression of active phosphogluconate dehydratase;
o) a disrupted endogenous glpK gene preventing expression of active glycerol kinase;
p) a disrupted endogenous gldA gene preventing expression of active NADH-dependent glycerol dehydrogenase; and
q) any one of the nucleotide sequences for a PSYCO construct SEQ ID NOs:65, 66, 67, or 68.
US10/680,286 2002-10-04 2003-10-06 Process for the biological production of 1,3-propanediol with high yield Expired - Lifetime US7371558B2 (en)

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